Morphogenic protein compositions of matter

ABSTRACT

Disclosed are novel compositions of morphogenic proteins constituting soluble forms of these proteins, antibodies that distinguish between soluble and mature forms, and method for producing these morphogenic proteins and antibodies.

This application is a continuation of application U.S. Ser. No.08/459,346, filed Jun. 2, 1995, now U.S. Pat. No. 5,834,179, which is adivisional of (1) copending application U.S. Ser. No. 08/402,542, filedon Mar. 13, 1995, which is a continuation of U.S. Ser. No. 08/040,510,filed Mar. 31, 1993, now abandoned; which is a continuation-in-part ofU.S. Ser. No. 08/029,335, filed Mar. 4, 1993, now abandoned; (2) U.S.Ser. No. 08/027,070, filed Mar. 4, 1993, now abandoned; (3) U.S. Ser.No. 07/971,091, filed Nov. 3, 1992, now abandoned; (4) U.S. Ser. No.07/946,235, filed Sep. 16, 1992, now abandoned; (5) U.S. Ser. No.07/938,336, filed Aug. 28, 1992, now abandoned; (6) U.S. Ser. No.07/923,780, filed Jul. 31, 1992, now abandoned, which is acontinuation-in-part of U.S. Ser. No. 07/752,857, filed Aug. 30, 1991,now abandoned; and (7) U.S. Ser. No. 07/752,764, filed Aug. 30, 1991,now abandoned, which is a continuation-in-part of U.S. Ser. No.07/667,274, filed Mar. 11, 1991, now abandoned. The disclosures of theseapplications are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates generally to morphogenic proteins and,more particularly, to compositions having improved solubility in aqueoussolvents.

BACKGROUND OF THE INVENTION

Morphogenic proteins ("morphogens") are well known and described in theart. See, for example, U.S. Pat. Nos. 4,968,590; 5,011,691; 5,018,753;PCT US92/01968 and PCT US92/07432; as well as various articles publishedin the scientific literature, including Ozkaynak et al. (1992) J. Biol.Chem. 267:25220-25227 and Ozkaynak et al. (1991) Biochem. Biophys. Res.Comm. 179:116-123. The art has described how to isolate morphogenicproteins from bone, how to identify genes encoding these proteins andhow to express them using recombinant DNA technology. The morphogenicproteins are capable of inducing endochondral bone formation and othertissue formation in a mammal when they are properly folded, dimerizedand disulfide bonded to produce a dimeric species having the appropriatethree dimensional conformation. The proteins have utility in therapeuticapplications, either by direct or systemic administration. Where boneinduction is desired, for example, the morphogen typically is providedto the desired site for bone formation in a mammal in association with asuitable matrix having the appropriate conformation to allow theinfiltration, proliferation and differentiation of migrating progenitorcells. The morphogenic protein adsorbed to the surfaces of a suitablematrix is generally referred to in the art as an osteogenic device. Theproteins can be isolated from bone or, preferably, the gene encoding theprotein is produced recombinantly in a suitable host cell.

The morphogen precursor polypeptide chains share a common structuralmotif, including a N-terminal signal sequence and pro region, both ofwhich are cleaved to produce a mature sequence, capable of disulfidebonding and comprising an N-terminal extension and a C-terminal domainwhose amino acid sequence is highly conserved among members of thefamily. In their mature dimeric forms, the morphogens typically arefairly insoluble under physiological conditions. Increasing thesolubility of these proteins has significant medical utility as it wouldenhance systemic administration of morphogens as therapeutics. Variouscarrier proteins, including serum albumin and casein are known toincrease the solubility of morphogens (see, for example, PCTUS92/07432). PCT US92/05309 (WO 93/00050) discusses the use of varioussolubilizing agents, including various amino acids and methyl estersthereof, as well as guanidine, sodium chloride and heparin, to increasethe solubility of mature dimeric BMP2.

Improved methods for the recombinant expression of morphogenic proteinsis an ongoing effort in the art. It is an object of this invention toprovide an improvement in the methods for producing and purifyingmorphogenic proteins having high specific activity, and for formulatingcompositions and osteogenic devices comprising these proteins. Anotherobject is to provide soluble forms of morphogenic proteins consistingessentially of amino acid sequences derived from morphogenic proteins.Another object is to provide formulations which stabilize the solublecomplex of morphogenic proteins. Still another object is to providemeans for distinguishing between soluble forms of the protein and themature morphogenic species, to provide means for quantitating theamounts of these proteins in a fluid, including a body fluid, such asserum, cerebro-sprinal fluid or peritoneal fluid, and to providepolyclonal and monoclonal antibodies capable of distinguishing betweenthese various species.

Another object is to provide antibodies and biological diagnostic assaysfor monitoring the concentration of morphogens and endogenousanti-morphogen antibodies present in a body fluid and to provide assaysfor detecting fluctuations in the concentrations of these proteins in abody fluid. U.S. Pat. No. 4,857,456 and Urist et al. (1984) Proc. Soc.Exp. Biol. Med. 176:472-475 describe a serum assay for detecting aprotein purported to be a bone morphogenetic protein. The protein is nota member of the morphogen family of proteins described herein, differingin molecular weight, structural characteristics and solubility fromthese proteins.

SUMMARY OF THE INVENTION

It has now been discovered that morphogenic protein secreted intocultured medium from mammalian cells contains as a significant fractionof the secreted protein a soluble form of the protein, and that thissoluble form comprises the mature dimeric species, including truncatedforms thereof, noncovalently associated with at least one, andpreferably two pro domains. It further has been discovered thatantibodies can be used to discriminate between these two forms of theprotein. These antibodies may be used as part of a purification schemeto selectively isolate the mature or the soluble form of morphogenicprotein, as well as to quantitate the amount of mature and soluble formsproduced. These antibodies also may be used as part of diagnostictreatments to monitor the concentration of morphogenic proteins insolution in a body and to detect fluctuations in the concentration ofthe proteins in their various forms. The antibodies and proteins alsomay be used in diagnostic assays to detect and monitor concentrations ofendogenous anti-morphogen antibodies to the various forms of theseproteins in the body.

An important embodiment of the invention is a dimeric protein comprisinga pair of polypeptide subunits associated to define a dimeric structurehaving morphogenic activity. As defined herein and in parent, relatedapplications, morphogens generally are capable of all of the followingbiological functions in a morphogenically permissive environment:stimulating proliferation of progenitor cells; stimulating thedifferentiation of progenitor cells; stimulating the proliferation ofdifferentiated cells; and supporting the growth and maintenance ofdifferentiated cells.

Each of the subunits of the dimeric morphogenic protein comprises atleast the 100 amino acid peptide sequence having the pattern of seven ormore cysteine residues characteristic of the morphogen family.Preferably, at least one of the subunits comprises the mature form of asubunit of a member of the morphogen family, or an allelic, species,mutant or chimeric variant thereof, noncovalently complexed with apeptide comprising part or all of a pro region of a member of themorphogen family, or an allelic, species, chimeric or other sequencevariant thereof. The pair of subunits and one or, preferably, two proregion peptides, together form a complex which is more soluble inaqueous solvents than the uncomplexed pair of subunits.

Preferably, both subunits comprise a mature form of a subunit of amember of the morphogen family or an allelic, species, chimeric, orother sequence variant thereof, and both subunits are noncovalentlycomplexed with a pro region comprising peptide, or a fragment thereof.Most preferably, each subunit is the mature form of human OP-1, or aspecies, allelic or other mutant variant thereof, and the pro region isthe entire or partial sequence of the pro region of human OP-1, or aspecies, allelic or other mutant variant thereof. Preferred pro regionsare full length forms of the pro region. Pro region fragments preferablyinclude the first 18 amino acids of the pro sequence. Other useful proregion fragments are truncated sequences of the intact pro regionsequence, the truncation occurring at the proteolytic cleavage siteArg-Xaa-Xaa-Arg (SEQ ID NO: 23).

As used herein, the mature form of a morphogen protein subunit includesthe intact C-terminal domain and intact or truncated forms of theN-terminal extensions. For example, useful mature forms of OP-1 includedimeric species defined by residues 293-431 of Seq ID No. 1, as well astruncated sequences thereof, including sequences defined by residues300-431, 313-431, 315-431, 316-431 and 318-431. Note that this lastsequence retains only about the last 10 residues of the N-terminalextension sequence. FIG. 2 presents the N-terminal extensions for anumber of preferred morphogen sequences. Canonical Arg-Xaa-Xaa-Arg (SEQID NO: 23) cleavage sites where truncation may occur are boxed orunderlined in the figure. As will be appreciated by those skilled in theart, mature dimeric species may include subunit combinations havingdifferent N-terminal truncations.

Other soluble forms of morphogens include dimers of the uncleaved proforms of these proteins (see below), as well as "hemi-dimers" whereinone subunit of the dimer is an uncleaved pro form of the protein, andthe other subunit comprises the mature form of the protein, includingtruncated forms thereof, preferably noncovalently associated with acleaved pro domain.

The soluble proteins of this invention are useful in the formation oftherapeutic compositions for administration to a mammal, particularly ahuman, and for the development of biological assays for monitoring theconcentration of these proteins and endogenous antibodies to theseproteins in body fluids, including, but not limited to, serum,cerebrospinal fluid and peritoneal fluid.

The foregoing and other objects, features and advantages of the presentinvention will be made more apparent from the following detaileddescription of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of a morphogen polypeptide chain asexpressed from a nucleic acid encoding the sequence, wherein thecross-hatched region represents the signal sequence; the stippled regionrepresents the pro domain; the hatched region represents the N-terminus("N-terminal extension") of the mature protein sequence; and the openregion represents the C-terminal region of the mature protein sequencedefining the conserved seven cysteine domain, the conserved cysteinesbeing indicated by vertical hatched lines;

FIG. 2 lists the sequences of the N-terminal extensions of the matureforms of various morphogens SEQ ID NO: 5, 1, 14, 18, 16, 12, 10, 11, 13,17, respectively, and

FIG. 3 is a gel filtration column elution profile of a soluble morphogen(OP-1) produced and purified from a mammalian cell culture by IMAC,S-Sepharose and S-200HR chromatography in TBS (Tris-buffered saline),wherein V_(o) is the void volume, ADH is alcohol dehydrogenase (MW 150kDa), BSA is bovine serum albumin (MW 67 kDa), CA is carbonic anhydrase(MW 29 kDa) and CytC is cytochrome C (MW 12.5 kDa).

DETAILED DESCRIPTION

A soluble form of morphogenic proteins now has been discovered whereinthe proteins consist essentially of the amino acid sequence of theprotein. The soluble form is a non-covalently associated complexcomprising the pro domain or a fragment thereof, noncovalentlyassociated or complexed with a dimeric protein species havingmorphogenic activity, each polypeptide of the dimer having less than 200amino acids and comprising at least the C-terminal six, and preferablyseven cysteine skeleton defined by residues 335-431 and 330-431respectively, of Seq. ID No. 1. Preferably, the polypeptide chains ofthe dimeric species comprise the mature forms of these sequences, ortruncated forms thereof. Preferred truncated forms comprise the intactC-terminal domain and at least 10 amino acids of the N-terminalextension sequence. The soluble forms of these morphogenic proteins maybe isolated from cultured cell medium, a mammalian body fluid, or may beformulated in vitro.

In vivo, under physiological conditions, the pro domain may serve toenhance the transportability of the proteins, and/or to protect theproteins from proteases and scavenger molecules, including antibodies.The pro domains also may aid in targeting the proteins to a particulartissue and/or to present the morphogen to a morphogen cell surfacereceptor by interaction with a co-receptor molecule. The isolatedproteins may be used in therapeutic formulations, particularly for oralor parenteral administration, and in the development of diagnosticassays to monitor the level of endogenous morphogens and endogenousanti-morphogen antibodies.

Detailed descriptions of the utility of these morphogens in therapies toregenerate lost or damaged tissues and/or to inhibit the tissuedestructive effects of tissue disorders or diseases, are provided inco-pending U.S. patent application Ser. No. 07/752,764, filed Aug. 31,1991 now abandoned in favor of continuation application U.S. Ser. No.08/404,113; U.S. Ser. No. 07/938,336, filed Aug. 28, 1992, now abandonedin favor of continuation application U.S. Ser. No. 08/445,467; U.S. Ser.No. 07/923,780, filed Jul. 31, 1992, now abandoned in favor ofcontinuation application U.S. Ser. No. 08/432,883; U.S. Ser. No.07/945,292, filed Sep. 15, 1992, now abandoned in favor of continuationapplication U.S. Ser. No. 08/271,556; U.S. Ser. No. 07/945,285, filedSep. 15, 1992, now abandoned in favor of continuation application U.S.Ser. No. 08/155,343; U.S. Ser. No. 07/938,337, filed Aug. 28, 1992, nowabandoned in favor of continuation application U.S. Ser. No. 08/480,515;U.S. Ser. No. 07/922,813, filed Jul. 31, 1992, now abandoned in favor ofcontinuation application U.S. Ser. No. 08/260,675; U.S. Ser. No.07/946,235, filed Sep. 16, 1992, now abandoned in favor of continuationapplication U.S. Ser. No. 08/278,730; U.S. Ser. No. 07/946,238, filedSep. 16, 1992, now abandoned in favor of continuation application U.S.Ser. No. 08/445,468; U.S. Ser. No. 07/945,286, filed Sep. 15, 1992, nowabandoned in favor of continuation application U.S. Ser. No. 08/461,397;and U.S. Ser. No. 07/971,071, filed Nov. 3, 1992, now abandoned in favorof continuation application U.S. Ser. No. 08/480,528, the disclosures ofwhich are incorporated herein by reference. Morphogens, including thesoluble morphogen complexes of this invention, are envisioned to haveparticular utility as part of therapies for regenerating lost or damagedbone, dentin, periodontal, liver, cardiac, lung and nerve tissue, aswell as for protecting these tissues from the tissue destructive effectsassociated with an immunological response. The proteins also areanticipated to provide a tissue protective effect in the treatment ofmetabolic bone disorders, such as osteoporosis, osteomalacia andosteosarcoma; in the treatment of liver disorders, including cirrhosis,hepatitis, alcohol liver disease and hepatic encephalopathy; and in thetreatment or prevention of ischemia reperfusion-associated tissuedamage, particularly to nerve or cardiac tissue.

Presented below are detailed descriptions of useful soluble morphogencomplexes of this invention, as well as how to make and use them.

I. Useful Soluble Morphogen Complexes--Protein Considerations

Among the morphogens useful in this invention are proteins originallyidentified as osteogenic proteins, such as the OP-1, OP-2 and CBMP2proteins, as well as amino acid sequence-related proteins such as DPP(from Drosophila), Vgl (from Xenopus), Vgr-1 (from mouse, see U.S. Pat.No. 5,011,691 to Oppermann et al.), GDF-1 (from mouse, see Lee (1991)PNAS 88:4250-4254), 60A protein (from Drosophila, see Wharton et al.(1991) PNAS 88:9214-9218), and the recently identified OP-3.

The members of this family, which are a subclass of the TGF-βsuper-family of proteins, share characteristic structural features,represented schematically in FIG. 1, as well as substantial amino acidsequence homology in their C-terminal domains, including a conservedseven cysteine structure. As illustrated in the figure, the proteins aretranslated as a precursor polypeptide sequence 10, having an N-terminalsignal peptide sequence 12, (the "pre pro" region, indicated in thefigure by cross-hatching), typically less than about 30 residues,followed by a "pro" region 14, indicated in the figure by stippling, andwhich is cleaved to yield the mature sequence 16. The mature sequencecomprises both the conserved C-terminal seven cysteine domain 20, and anN-terminal sequence 18, referred to herein as an N-terminal extension,and which varies significantly in sequence between the variousmorphogens. Cysteines are represented in the figure by vertical hatchedlines 22. The polypeptide chains dimerize and these dimers typically arestabilized by at least one interchain disulfide bond linking the twopolypeptide chain subunits.

The signal peptide is cleaved rapidly upon translation, at a cleavagesite that can be predicted in a given sequence using the method of VonHeijne ((1986) Nucleic Acids Research 14:4683-4691.) The "pro" form ofthe protein subunit, 24, in FIG. 1, includes both the pro domain and themature domain, peptide bonded together. Typically, this pro form iscleaved while the protein is still within the cell, and the pro domainremains noncovalently associated with the mature form of the subunit toform a soluble species that appears to be the primary form secreted fromcultured mammalian cells. Typically, previous purification techniquesutilized denaturing conditions that disassociated the complex.

Other soluble forms of morphogens secreted from mammalian cells includedimers of the pro forms of these proteins, wherein the pro region is notcleaved from the mature domain, and "hemi-dimers", wherein one subunitcomprises a pro form of the polypeptide chain subunit and the othersubunit comprises the cleaved mature form of the polypeptide chainsubunit (including truncated forms thereof), preferably noncovalentlyassociated with a cleaved pro domain.

The isolated pro domain typically has a substantial hydrophobiccharacter, as determined both by analysis of the sequence and bycharacterization of its properties in solution. The isolated pro regionsalone typically are not significantly soluble in aqueous solutions, andrequire the presence of denaturants, e.g., detergents, urea, guanidineHCl, and the like, and/or one or more carrier proteins. Accordingly,without being limited to any given theory, the non-covalent associationof the cleaved pro region with the mature morphogen dimeric specieslikely involves interaction of a hydrophobic portion of the pro regionwith a corresponding hydrophobic region on the dimeric species, theinteraction of which effectively protects or "hides" an otherwiseexposed hydrophobic region of the mature dimer from exposure to aqueousenvironments, enhancing the affinity of the mature dimer species foraqueous solutions.

Morphogens comprise a subfamily of proteins within the TGF-β superfamilyof structurally related proteins. Like the morphogens described herein,TGF-β also has a pro region which associates non-covalently with themature TGF-β protein form. However, unlike the morphogens, the TGF-β proregion contains numerous cysteines and forms disulfide bonds with aspecific binding protein. The TGF-β1 pro domain also is phosphorylatedat one or more mannose residues, while the morphogen pro regionstypically are not.

Useful pro domains include the full length pro regions described below,as well as various truncated forms hereof, particularly truncated formscleaved at proteolytic Arg-Xaa-Xaa-Arg (SEQ ID NO: 23) cleavage sites.For example, in OP-1, possible pro sequences include sequences definedby residues 30-292 (full length form); 48-292; and 158-292. Soluble OP-1complex stability is enhanced when the pro region comprises the fulllength form rather than a truncated form, such as the 48-292 truncatedform, in that residues 30-47 show sequence homology to the N-terminalportions of other morphogens, and are believed to have particularutility in enhancing complex stability for all morphogens. Accordingly,currently preferred pro sequences are those encoding the full lengthform of the pro region for a given morphogen (see below). Other prosequences contemplated to have utility include biosynthetic prosequences, particularly those that incorporate a sequence derived fromthe N-terminal portion of one or more morphogen pro sequences.

Table I, below, describes the various preferred morphogens identified todate, including their nomenclature as used herein, the sequencesdefining the various regions of the subunit sequences, their Seq. IDreferences, and publication sources for their nucleic acid and aminoacid sequences. The disclosure of these publications is incorporatedherein by reference. The mature protein sequences defined are thelongest anticipated forms of these sequences. As described above,shorter, truncated forms of these sequences also are contemplated.Preferably, truncated mature sequences include at least 10 amino acidsof the N-terminal extension. FIG. 2 lists the N-terminal extensions fora number of the preferred morphogen sequences described below.Arg-Xaa-Xaa-Arg (SEQ ID NO: 23) cleavage sites that may yield truncatedsequences of the mature subunit form are boxed or underlined in thefigure.

TABLE I

"OP-1" Refers generically to the group of morphogenically activeproteins expressed from part or all of a DNA sequence encoding OP-1protein, including allelic and species variants thereof, e.g., humanOP-1 ("hOP-1"), or mouse OP-1 ("mOP-1".) The cDNA sequences and theamino acids encoding the full length proteins are provided in Seq. IdNos. 1 and 2 (hOP1) and Seq. ID Nos. 3 and 4 (mOP1.) The mature proteinsare defined by residues 293-431 (hOP1) and 292-430 (mOP1), wherein theconserved seven cysteine skeleton is defined by residues 330-431 and329-430, respectively, and the N-terminal extensions are defined byresidues 293-329 and 292-329, respectively. The "pro" regions of theproteins, cleaved to yield the mature, morphogenically active proteins,are defined essentially by residues 30-292 (hOP1) and residues 30-291(mOP1).

"OP-2" refers generically to the group of active proteins expressed frompart or all of a DNA sequence encoding OP-2 protein, including allelicand species variants thereof, e.g., human OP-2 ("hOP-2") or mouse OP-2("mOP-2".) The full length proteins are provided in Seq. ID Nos. 5 and 6(hOP2) and Seq. ID Nos. 7 and 8 (mOP2.) The mature proteins are definedessentially by residues 264-402 (hOP2) and 261-399 (mOP2), wherein theconserved seven cysteine skeleton is defined by residues 301-402 and298-399, respectively, and the N-terminal extensions are defined byresidues 264-300 and 261-297, respectively. The "pro" regions of theproteins, cleaved to yield the mature, morphogenically active proteinslikely are defined essentially by residues 18-263 (hOP2) and residues18-260 (mOP2). (Another cleavage site also occurs 21 residues upstreamfor both OP-2 proteins.)

"OP-3" refers generically to the group of active proteins expressed frompart or all of a DNA sequence encoding OP-3 protein, including allelicand species variants thereof, e.g., mouse OP-3 ("mOP-3".) The fulllength protein is provided in Seq. ID No. 9. The mature protein isdefined essentially by residues 261-399 or 264-399, wherein theconserved seven cysteine skeleton is defined by residues 298-399 and theN-terminal extension is defined by residues 264-297 or 261-297. The"pro" region of the protein, cleaved to yield the mature,morphogenically active proteins likely is defined essentially byresidues 20-262.

"BMP2/BMP4" refers to protein sequences encoded by the human BMP2 andBMP4 genes. The amino acid sequence for the full length proteins,referred to in the literature as BMP2A and BMP2B, or BMP2 and BMP4,appear in Seq. ID Nos. 10 and 11, respectively, and in Wozney, et al.(1988) Science 242:1528-1534. The pro domain for BMP2 (BMP2A) likelyincludes residues 25-248 or 25-282; the mature protein, residues 249-396or 283-396, of which residues 249-296/283-296 define the N-terminalextension and 295-396 define the C-terminal domain. The pro domain forBMP4 (BMP2B) likely includes residues 25-256 or 25-292; the matureprotein, residues 257-408 or 293-408, of which 257-307/293-307 definethe N-terminal extension, and 308-408 define the C-terminal domain.

"DPP" refers to protein sequences encoded by the Drosophila DPP gene.The amino acid sequence for the full length protein, including themature form and the pro region, appears in Seq. ID No. 12 and inPadgett, et al (1987) Nature 325: 81-84. The pro domain likely extendsfrom the signal peptide cleavage site to residue 456; the mature proteinlikely is defined by residues 457-588, where residues 457-586 define theN-terminal extension and 487-588 define the C-terminal domain.

"Vgl" refers to protein sequences encoded by the Xenopus Vgl gene. Theamino acid sequence for the full length protein, including the matureform and the pro region, appears in Seq.ID No. 13 and in Weeks (1987)Cell 51: 861-867. The pro domain likely extends from the signal peptidecleavage site to residue 246; the mature protein likely is defined byresidues 247-360, where residues 247-258 define the N-terminalextension, and residues 259-360 define the C-terminal domain.

"Vgr-1" refers to protein sequences encoded by the murine Vgr-1 gene.The amino acid sequence for the full length protein, including themature form and the pro region, appears in Seq. ID No. 14 and in Lyons,et al, (1989) PNAS 86: 4554-4558. The pro domain likely extends from thesignal peptide cleavage site to residue 299; the mature protein likelyis defined by residues 300-438, where residues 300-336 define theN-terminal extension and residues 337-438 define the C-terminus.

"GDF-1" refers to protein sequences encoded by the human GDF-1 gene. ThecDNA and encoded amino sequence for the full length protein is providedin Seq. ID. No. 15 and Lee (1991) PNAS 88:4250-4254. The pro domainlikely extends from the signal peptide cleavage site to residue 214; themature protein likely is defined by residues 215-372, where residues215-256 define the N-terminal extension and residues 257-372 define theC-terminus.

"60A" refers to protein sequences encoded by the Drosophila 60A gene.The amino acid sequence for the full length protein appears in Seq. IDNo. 16 and in Wharton et al. (1991) PNAS 88:9214-9218) The pro domainlikely extends from the signal peptide cleavage site to residue 324; themature protein likely is defined by residues 325-455, wherein residues325-353 define the N-terminal extension and residues 354-455 define theC-terminus.

"BMP3" refers to protein sequences encoded by the human BMP3 gene. Theamino acid sequence for the full length protein, including the matureform and the pro region, appears in Seq.ID No. 17 and in Wozney et al.(1988) Science 242: 1528-1534. The pro domain likely extends from thesignal peptide cleavage site to residue 290; the mature protein likelyis defined by residues 291-472, wherein residues 291-370 define theN-terminal extension and residues 371-472 define the C-terminus.

"BMP5" refers to protein sequences encoded by the human BMP5 gene. Theamino acid sequence for the full length protein, including the matureform and the pro region, appears in Seq.ID No. 18 and in Celeste, et al.(1990) PNAS 87: 9843-9847. The pro domain likely extends from the signalpeptide cleavage site to residue 316; the mature protein likely isdefined by residues 317-454, where residues 317-352 define theN-terminus and residues 352-454 define the C-terminus.

"BMP6" refers to protein sequences encoded by the human BMP6 gene. Theamino acid sequence for the full length protein, including the matureform and the pro region, appears in Seq. ID No. 19 and in Celeste, etal. (1990) PNAS 87: 9843-5847. The pro domain likely includes extendsfrom the signal peptide cleavage site to residue 374; the maturesequence likely includes residues 375-513, where residues 375-411 definethe N-terminus and residues 412-513 define the C-terminus.

Note that the OP-2 and OP-3 proteins have an additional cysteine residuein the C-terminal region (e.g., see residue (341 in SEQ ID NO: 5), inaddition to the conserved cysteine skeleton in common with the otherproteins in this family. The GDF-1 protein has a four amino acid insertwithin the conserved skeleton ("Gly-Gly-Pro-Pro") but this insert likelydoes not interfere with the relationship of the cysteines in the foldedstructure. In addition, the CBMP2 proteins are missing one amino acidresidue within the cysteine skeleton.

The dimeric morphogen species are inactive when reduced, but are activeas oxidized homodimers and when oxidized in combination with othermorphogens of this invention. Thus, as defined herein, a morphogenuseful in a soluble morphogen complex is a dimeric protein comprising apair of polypeptide chains, wherein each polypeptide chain has less than200 amino acids and comprises at least the C-terminal six, preferablyseven cysteine skeleton defined by residues 335-431 of Seq. ID No. 1,including functionally equivalent arrangements of these cysteines (e.g.,amino acid insertions or deletions which alter the linear arrangement ofthe cysteines in the sequence but not their relationship in the foldedstructure), such that, when the polypeptide chains are folded, thedimeric protein species comprising the pair of polypeptide chains hasthe appropriate three-dimensional structure, including the appropriateintra- or inter-chain disulfide bonds such that the protein is capableof acting as a morphogen as defined herein. The solubility of thesestructures is improved when the mature dimeric form of a morphogen, inaccordance with the invention, is complexed with at least one, andpreferably two, pro domains.

Various generic sequences (Generic Sequence 1-6) defining preferredC-terminal sequences useful in the soluble morphogens of this inventionare described in U.S. Ser. No. 07/923,780, now abandoned in favor ofcontinuation application U.S. Ser. No. 08/432,883, incorporated hereinabove by reference. Two currently preferred generic sequences aredescribed below.

Generic Sequence 7 (Seq. ID No. 20) and Generic Sequence 8 (Seq. ID No.21) disclosed below, accommodate the homologies shared among preferredmorphogen protein family members identified to date, including OP-1,OP-2, OP-3, CBMP2A, CBMP2B, BMP3, 60A, DPP, Vgl, BMP5, BMP6, Vrg-1, andGDF-1. The amino acid sequences for these proteins are described herein(see Sequence Listing) and/or in the art, as well as in PCT publicationU.S. Ser. No. 92/07358 (WO93/04692), filed Aug. 28, 1992, for example.The generic sequences include both the amino acid identity shared bythese sequences in the C-terminal domain, defined by the six and sevencysteine skeletons (Generic Sequences 7 and 8, respectively), as well asalternative residues for the variable positions within the sequence. Thegeneric sequences allow for an additional cysteine at position 41(Generic Sequence 7) or position 46 (Generic Sequence 8), providing anappropriate cysteine skeleton where inter- or intramolecular disulfidebonds can form, and containing certain critical amino acids whichinfluence the tertiary structure of the proteins.

    Generic Sequence 7                                                                 Leu Xaa Xaa Xaa Phe                                                             1               5                                                      Xaa Xaa Xaa Gly Trp Xaa Xaa Xaa Xaa                                                            10                                                           Xaa Xaa Pro Xaa Xaa Xaa Xaa Ala                                                15                  20                                                       Xaa Tyr Cys Xaa Gly Xaa Cys Xaa                                                       25                  30                                                Xaa Pro Xaa Xaa Xaa Xaa Xaa                                                                    35                                                           Xaa Xaa Xaa Asn His Ala Xaa Xaa                                                       40                  45                                                Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa                                                                50                                                           Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys                                                    55                  60                                                   Cys Xaa Pro Xaa Xaa Xaa Xaa Xaa                                                            65                                                               Xaa Xaa Xaa Leu Xaa Xaa Xaa                                                    70                  75                                                       Xaa Xaa Xaa Xaa Val Xaa Leu Xaa                                                            80                                                               Xaa Xaa Xaa Xaa Met Xaa Val Xaa                                                85                  90                                                       Xaa Cys Xaa Cys Xaa                                                                    95                                                               

wherein each Xaa is independently selected from a group of one or morespecified amino acids defined as follows: "Res." means "residue" and Xaaat res.2=(Tyr or Lys); Xaa at res.3=Val or Ile); Xaa at res.4=(Ser, Aspor Glu); Xaa at res.6=(Arg, Gln, Ser, Lys or Ala); Xaa at res.7=(Asp orGlu); Xaa at res.8=(Leu, Val or Ile); Xaa at res.11=(Gln, Leu, Asp, His,Asn or Ser); Xaa at res.12=(Asp, Arg, Asn or Glu); Xaa at res. 13=(Trpor Ser); Xaa at res.14=(Ile or Val); Xaa at res.15=(Ile or Val); Xaa atres.16 (Ala or Ser); Xaa at res.18=(Glu, Gln, Leu, Lys, Pro or Arg); Xaaat res.19=(Gly or Ser); Xaa at res.20=(Tyr or Phe); Xaa at res.21=(Ala,Ser, Asp, Met, His, Gln, Leu or Gly); Xaa at res.23=(Tyr, Asn or Phe);Xaa at res.26=(Glu, His, Tyr, Asp, Gln, Ala or Ser); Xaa at res.28=(Glu,Lys, Asp, Gln or Ala); Xaa at res.30=(Ala, Ser, Pro, Gln, Ile or Asn);Xaa at res.31=(Phe, Leu or Tyr); Xaa at res.33=(Leu, Val or Met); Xaa atres.34=(Asn, Asp, Ala, Thr or Pro); Xaa at res.35=(Ser, Asp, Glu, Leu,Ala or Lys); Xaa at res.36=(Tyr, Cys, His, Ser or Ile); Xaa atres.37=(Met, Phe, Gly or Leu); Xaa at res.38=(Asn, Ser or Lys); Xaa atres.39=(Ala, Ser, Gly or Pro); Xaa at res.40=(Thr, Leu or Ser); Xaa atres.44=(Ile, Val or Thr); Xaa at res.45=(Val, Leu, Met or Ile); Xaa atres.46=(Gln or Arg); Xaa at res.47=(Thr, Ala or Ser); Xaa at res.48=(Leuor Ile); Xaa at res.49=(Val or Met); Xaa at res.50=(His, Asn or Arg);Xaa at res.51=(Phe, Leu, Asn, Ser, Ala or Val); Xaa at res.52=(Ile, Met,Asn, Ala, Val, Gly or Leu); Xaa at res.53=(Asn, Lys, Ala, Glu, Gly orPhe); Xaa at res.54=(Pro, Ser or Val); Xaa at res.55=(Glu, Asp, Asn,Gly, Val, Pro or Lys); Xaa at res.56=(Thr, Ala, Val, Lys, Asp, Tyr, Ser,Gly, Ile or His); Xaa at res.57=(Val, Ala or Ile); Xaa at res.58=(Pro orAsp); Xaa at res.59=(Lys, Leu or Glu); Xaa at res.60=(Pro, Val or Ala);Xaa at res.63=(Ala or Val); Xaa at res.65=(Thr, Ala or Glu); Xaa atres.66=(Gln, Lys, Arg or Glu); Xaa at res.67=(Leu, Met or Val); Xaa atres.68=(Asn, Ser, Asp or Gly); Xaa at res.69=(Ala, Pro or Ser); Xaa atres.70=(Ile, Thr, Val or Leu); Xaa at res.71=(Ser, Ala or Pro); Xaa atres.72=(Val, Leu, Met or Ile); Xaa at res.74=(Tyr or Phe); Xaa atres.75=(Phe, Tyr, Leu or His); Xaa at res.76=(Asp, Asn or Leu); Xaa atres.77=(Asp, Glu, Asn, Arg or Ser); Xaa at res.78=(Ser, Gln, Asn, Tyr orAsp); Xaa at res.79=(Ser, Asn, Asp, Glu or Lys); Xaa at res.80=(Asn, Thror Lys); Xaa at res.82=(Ile, Val or Asn); Xaa at res.84=(Lys or Arg);Xaa at res.85=(Lys, Asn, Gln, His, Arg or Val); Xaa at res.86=(Tyr, Gluor His); Xaa at res.87=(Arg, Gln, Glu or Pro); Xaa at res.88=(Asn, Glu,Trp or Asp); Xaa at res.90=(Val, Thr, Ala or Ile); Xaa at res.92=(Arg,Lys, Val, Asp, Gln or Glu); Xaa at res.93=(Ala, Gly, Glu or Ser); Xaa atres.95=(Gly or Ala) and Xaa at res.97=(His or Arg).

As described above, Generic Sequence 8 (Seq. ID No. 21) includes all ofGeneric Sequence 7 and in addition includes the following sequence atits N-terminus: Cys Xaa Xaa Xaa Xaa 1 5

Accordingly, beginning with residue 7, each "Xaa" in Generic Seq. 8 is aspecified amino acid defined as for Generic Seq. 7, with the distinctionthat each residue number described for Generic Sequence 7 is shifted byfive in Generic Seq. 8. Thus, "Xaa at res.2=(Tyr or Lys)" in Gen. Seq. 7refers to Xaa at res. 7 in Generic Seq. 8. In Generic Seq. 8, Xaa atres.2=(Lys, Arg, Ala or Gln); Xaa at res.3=(Lys, Arg or Met); Xaa atres.4=(His, Arg or Gln); and Xaa at res.5=(Glu, Ser, His, Gly, Arg, Pro,Thr, or Tyr).

Accordingly, other useful sequences defining preferred C-terminalsequences are those sharing at least 70% amino acid sequence homology or"similarity", and preferably 80% homology or similarity with any of thesequences incorporated into Generic Seq. 7 and 8 above. These areanticipated to include allelic, species other sequence amino acidsequence variants, as well as novel members of this morphogenic familyof proteins. As used herein, "amino acid sequence homology" isunderstood to mean amino acid sequence similarity, and homologoussequences share identical or similar amino acids, where similar aminoacids are conserved amino acids as defined by Dayoff et al., Atlas ofProtein Sequence and Structure; vol.5, Suppl.3, pp.345-362 (M. O.Dayoff, ed., Nat'l BioMed. Research Fdn., Washington D.C. 1978.) Thus, acandidate sequence sharing 70% amino acid homology with a referencesequence requires that, following alignment of the candidate sequencewith the reference sequence, 70% of the amino acids in the candidatesequence are identical to the corresponding amino acid sequences in thereference sequence, or constitute a conserved amino acid change thereto."Amino acid sequence identity" is understood to require identical aminoacids between two aligned sequences. Thus, a candidate sequence sharing60% amino acid identity with a reference sequence requires that,following alignment of the candidate sequence with the referencesequence, 60% of the amino acids in the candidate sequence are identicalto the corresponding amino acid in the reference sequence.

As used herein, all homologies and identities calculated use OP-1 as thereference sequence. Also as used herein, sequences are aligned forhomology and identity calculations using the method of Needleman et al.(1970) J. Mol. Biol. 48:443-453 and identities calculated by the Alignprogram (DNAstar, Inc.) In all cases, internal gaps and amino acidinsertions in the candidate sequence as aligned are ignored when makingthe homology/identity calculation.

Also as used herein, "sequence variant" is understood to mean an aminoacid variant form of the morphogen protein, wherein the amino acidchange or changes in the sequence do not alter significantly themorphogenic activity (e.g., tissue regeneration activity). of theprotein, and the variant molecule performs substantially the samefunction in substantially the same way as the naturally occurring formof the molecule. Sequence variants may include single or multiple aminoacid changes, and are intended to include chimeric sequences asdescribed below. The variants may be naturally occurring or may bebiosynthetically induced by using standard recombinant DNA techniques orchemical protein synthesis methodologies.

The currently most preferred protein sequences useful in solublemorphogen complexes in this invention include those having greater than60% identity, preferably greater than 65% identity, with the amino acidsequence defining the conserved six cysteine skeleton of hOP1 (e.g.,residues 335-431 of Seq. ID No. 1). These most preferred sequencesinclude both allelic and species variants of the OP-1 and OP-2 proteins,including the Drosophila 60A protein. Accordingly, in another preferredaspect of the invention, useful morphogens include active proteinscomprising species of polypeptide chains having the generic amino acidsequence herein referred to as "OPX", which accommodates the homologiesbetween the various identified species of OP1 and OP2 (Seq. ID No. 22).

Useful N-terminal extension sequences are listed in FIG. 2 for use withthe C-terminal domains described above. Also as described above, thefull length N-terminal extensions, or truncated forms thereof, may beused in preferred dimeric species. The mature dimeric species may beproduced from intact DNAs, or truncated forms thereof. It also isenvisioned as an embodiment of the invention that chimeric morphogensequences can be used. Thus, DNAs encoding chimeric morphogens may beconstructed using part or all of N-terminal extension from one morphogenand a C-terminal domain derived from one or more other morphogens. Thesechimeric proteins may be synthesized using standard recombinant DNAmethodology and/or automated chemical nucleic acid synthesis methodologywell described in the art. Other chimeric morphogens include solublemorphogen complexes where the pro domain is encoded from a DNA sequencecorresponding to one morphogen, and part or all of the mature domain isencoded by DNA derived from other, different morphogen(s). These solublechimerics may be produced from a single synthetic DNA as describedbelow, or, alternatively, may be formulated in vitro from isolatedcomponents also as described herein below.

Finally, the morphogen pro domains and/or mature form N-terminalextensions themselves may be useful as tissue targeting sequences. Asdescribed above, the morphogen family members share significant sequencehomology in their C-terminal active domains. By contrast, the sequencesdiverge significantly in the sequences which define the pro domain andthe N-terminal 39 amino acids of the mature protein. Accordingly, thepro domain and/or N-terminal extension sequence may bemorphogen-specific. Accordingly, part or all of these morphogen-specificsequences may serve as tissue targeting sequences for the morphogensdescribed herein. For example, the N-terminal extension and/or prodomains may interact specifically with one or more molecules at thetarget tissue to direct the morphogen associated with the pro domain tothat tissue. Thus, for example, the morphogen-specific sequences ofOP-1, BMP2 or BMP4, all of which proteins are found naturally associatedwith bone tissue (see, for example, U.S. Pat. No. 5,011,691) may beparticularly useful sequences when the morphogen complex is to betargeted to bone. Similarly, BMP6 (or Vgr-1) specific sequences may beused when targeting to lung tissue is desired. Alternatively, themorphogen-specific sequences of GDF-1 may be used to target solublemorphogen complexes to nerve tissue, particularly brain tissue, whereGDF-1 appears to be primarily expressed (see, for example, U.S. Ser. No.922,813 now abandoned in favor of continuation application U.S. Ser. No.08/260,675, and Lee, PNAS, 88:4250-4254 (1991), incorporated herein byreference).

II. Recombinant Production of Soluble Morphogen Complexes

Soluble morphogen complexes can be produced from eukaryotic host cells,preferably mammalian cells, using standard recombinant expressiontechniques. An exemplary protocol currently preferred, is providedbelow, using a particular vector construct and chinese hamster ovary(CHO) cell line. Those skilled in the art will appreciate that otherexpression systems are contemplated to be useful, including othervectors and other cell systems, and the invention is not intended to belimited to soluble morphogenic protein complexes produced only by themethod detailed hereinbelow. Similar results to those described hereinhave been observed using recombinant expression systems developed forCOS and BSC cells.

Morphogen DNA encoding the precursor sequence is subcloned into aninsertion site of a suitable, commercially available pUC-type vector(e.g., pUC-19, ATCC #37254, Rockville, Md.), along with a suitablepromoter/enhancer sequences and 3' termination sequences. Useful DNAsequences include the published sequences encoding these proteins,and/or synthetic constructs. Currently preferred promoter/enhancersequences are the CMV promoter (human cytomegalovirus majorintermediate--early promoter) and the mouse mammary tumor virus promoter(mMTV) boosted by the rous sarcoma virus LTR enhancer sequence (e.g.,from Clontech, Inc., Palo Alto). Expression also may be further enhancedusing transactivating enhancer sequences. The plasmid also contains DHFRas an amplifiable marker, under SV40 early promoter control (ATCC#37148). Transfection, cell culturing, gene amplification and proteinexpression conditions are standard conditions, well known in the art,such as are described, for example in Ausubel et al., ed., CurrentProtocols in Molecular Biology, John Wiley & Sons, NY (1989). Briefly,transfected cells are cultured in medium containing 0.1-0.5% dialyzedfetal calf serum (FCS) and stably transfected high expression cell linesare obtained by subcloning and evaluated by standard Western or Northernblot. Southern blots also are used to assess the state of integratedsequences and the extent of their copy number amplification.

A currently preferred expression vector contains the DHFR gene, underSV40 early promoter control, as both a selection marker and as aninducible gene amplifier. The DNA sequence for DHFR is wellcharacterized in the art, and is available commercially. For example, asuitable vector may be generated from pMAM-neo (Clontech, Inc., PaloAlto, Calif.) by replacing the neo gene (BamHI digest) with anSphI-BamHI, or a PvuII-BamHI fragment from pSV5-DHFR (ATCC #37148),which contains the DHFR gene under SV40 early promoter control. A BamHIsite can be engineered at the SphI or PvuII site using standardtechniques (e.g., by linker insertion or site-directed mutagenesis) toallow insertion of the fragment into the vector backbone. The morphogenDNA can be inserted into the polylinker site downstream of the MMTV-LTRsequence (mouse mammary tumor virus LTR). The CMV promoter sequence thenmay be inserted into the expression vector (e.g., from pCDM8,Invitrogen, Inc.) The SV40 early promoter, which drives DHFR expression,preferably is modified in these vectors to reduce the level of DHFR mRNAproduced.

The currently preferred mammalian cell line is a CHO Chinese hamsterovary, cell line, and the preferred procedure for establishing a stablemorphogen production cell line with high expression levels comprisestransfecting a stable CHO cell line, preferably CHO-DXB11, with theexpression vector described above, isolating clones with high morphogenexpression levels, and subjecting these clones to cycles of subcloningusing a limited dilution method described below to obtain a populationof high expression clones. Subcloning preferably is performed in theabsence of MTX to identify stable high expression clones which do notrequire addition of MTX to the growth media for morphogen production.

In the subcloning protocol cells are seeded on ten 100 mm petri dishesat a cell density of either 50 or 100 cells per plate, with orpreferably without MTX in the culture media. After 14 days of growth,clones are isolated using cloning cylinders and standard procedures, andcultured in 24-well plates. Clones then are screened for morphogenexpression by Western immunoblots using standard procedures, andmorphogen expression levels compared to parental lines. Cell linestability of high expression subclones then is determined by monitoringmorphogen expression levels over multiple cell passages (e.g., four orfive passages).

III. Isolation of Soluble Morphogen Complex from Conditioned Media orBody Fluid

Morphogens are expressed from mammalian cells as soluble complexes.Typically, however the complex is disassociated during purification,generally by exposure to denaturants often added to the purificationsolutions, such as detergents, alcohols, organic solvents, chaotropicagents and compounds added to reduce the pH of the solution. Providedbelow is a currently preferred protocol for purifying the solubleproteins from conditioned media (or, optionally, a body fluid such asserum, cerebro-spinal or peritoneal fluid), under non-denaturingconditions. The method is rapid, reproducible and yields isolatedsoluble morphogen complexes in substantially pure form.

Soluble morphogen complexes can be isolated from conditioned media usinga simple, three step chromatographic protocol performed in the absenceof denaturants. The protocol involves running the media (or body fluid)over an affinity column, followed by ion exchange and gel filtrationchromatographies. The affinity column described below is a Zn-IMACcolumn. The present protocol has general applicability to thepurification of a variety of morphogens, all of which are anticipated tobe isolatable using only minor modifications of the protocol describedbelow. An alternative protocol also envisioned to have utility animmunoaffinity column, created using standard procedures and, forexample, using antibody specific for a given morphogen pro domain(complexed, for example, to a protein A-conjugated Sepharose column.)Protocols for developing immunoaffinity columns are well described inthe art, (see, for example, Guide to Protein Purification, M. Deutscher,ed., Academic Press, San Diego, 1990, particularly sections VII and XI.)

In this experiment OP-1 was expressed in CHO cells as described above.The CHO cell conditioned media containing 0.5% FBS was initiallypurified using Immobilized Metal-Ion Affinity Chromatography (IMAC). Thesoluble OP-1 complex from conditioned media binds very selectively tothe Zn-IMAC resin and a high concentration of imidazole (50 mMimidazole, pH 8.0) is required for the effective elution of the boundcomplex. The Zn-IMAC step separates the soluble OP-1 from the bulk ofthe contaminating serum proteins that elute in the flow through and 35mM imidazole wash fractions. The Zn-IMAC purified soluble OP-1 is nextapplied to an S-Sepharose cation-exchange column equilibrated in 20 mMNaPO₄ (pH 7.0) with 50 mM NaCl. This S-Sepharose step serves to furtherpurify and concentrate the soluble OP-1 complex in preparation for thefollowing gel filtration step. The protein was applied to a SephacrylS-200HR column equilibrated in TBS. Using substantially the sameprotocol, soluble morphogens also may be isolated from one or more bodyfluids, including serum, cerebro-spinal fluid or peritoneal fluid.

IMAC was performed using Chelating-Sepharose (Pharmacia) that had beencharged with three column volumes of 0.2 M ZnSO₄. The conditioned mediawas titrated to pH 7.0 and applied directly to the ZN-IMAC resinequilibrated in 20 mM HEPES (pH 7.0) with 500 mM NaCl. The Zn-IMAC resinwas loaded with 80 mL of starting conditioned media per mL of resin.After loading the column was washed with equilibration buffer and mostof the contaminating proteins were eluted with 35 mM imidazole (pH 7.0)in equilibration buffer. The soluble OP-1 complex is then eluted with 50mM imidazole (pH 8.0) in 20 mM HEPES and 500 mM NaCl.

The 50 mM imidazole eluate containing the soluble OP-1 complex wasdiluted with nine volumes of 20 mM NaPO₄ (pH 7.0) and applied to anS-Sepharose (Pharmacia) column equilibrated in 20 mM NaPO₄ (pH 7.0) with50 mM NaCl. The S-Sepharose resin was loaded with an equivalent of 800mL of starting conditioned media per mL of resin. After loading theS-Sepharose column was washed with equilibration buffer and eluted with100 mM NaCl followed by 300 mM and 500 mM NaCl in 20 mM NaPO₄ (pH 7.0).The 300 mM NaCl pool was further purified using gel filtrationchromatography. Fifty mls of the 300 mm NaCl eluate was applied to a5.0×90 cm Sephacryl S-200HR (Pharmacia) equilibrated in Tris bufferedsaline (TBS), 50 mM Tris, 150 mM NaCl (pH 7.4). The column was eluted ata flow rate of 5 mL/minute collecting 10 mL fractions. The apparentmolecular of the soluble OP-1 was determined by comparison to proteinmolecular weight standards (alcohol dehydrogenase (ADH, 150 kDa), bovineserum albumin (BSA, 68 kDa), carbonic anhydrase (CA, 30 kDa) andcytochrome C (cyt C, 12.5 kDa). (see FIG. 3) The purity of the S-200column fractions was determined by separation on standard 15%polyacrylamide SDS gels stained with coomassie blue. The identity of themature OP-1 and the pro-domain was determined by N-terminal sequenceanalysis after separation of the mature OP-1 from the pro-domain usingstandard reverse phase C18 HPLC.

FIG. 3 shows the absorbance profile at 280 nm. The soluble OP-1 complexelutes with an apparent molecular weight of 110 kDa. This agrees wellwith the predicted composition of the soluble OP-1 complex with onemature OP-1 dimer (35-36 kDa) associated with two pro-domains (39 kDaeach). Purity of the final complex can be verified by running theappropriate fraction in a reduced 15% polyacrylamide gel.

The complex components can be verified by running the complex-containingfraction from the S-200 or S-200HR columns over a reverse phase C18 HPLCcolumn and eluting in an acetonitrile gradient (in 0.1% TFA), usingstandard procedures. The complex is dissociated by this step, and thepro domain and mature species elute as separate species. These separatespecies then can be subjected to N-terminal sequencing using standardprocedures (see, for example, Guide to Protein Purification, M.Deutscher, ed., Academic Press, San Diego, 1990, particularly pp.602-613), and the identity of the isolated 36 kD, 39 kDa proteinsconfirmed as mature morphogen and isolated, cleaved pro domain,respectively. N-terminal sequencing of the isolated pro domain frommammalian cell produced OP-1 revealed 2 forms of the pro region, theintact form (beginning at residue 30 of Seq. ID No. 1) and a truncatedform, (beginning at residue 48 of Seq. ID No. 1.) N-terminal sequencingof the polypeptide subunit of the isolated mature species reveals arange of N-termini for the mature sequence, beginning at residues 293,300, 313, 315, 316, and 318, of Seq. ID No. 1, all of which are activeas demonstrated by the standard bone induction assay.

V. In Vitro Soluble Morphogen Complex Formation

As an alternative to purifying soluble complexes from culture media or abody fluid, soluble complexes may be formulated from purified prodomains and mature dimeric species. Successful complex formationapparently requires association of the components under denaturingconditions sufficient to relax the folded structure of these molecules,without affecting disulfide bonds. Preferably, the denaturing conditionsmimic the environment of an intracellular vesicle sufficiently such thatthe cleaved pro domain has an opportunity to associate with the maturedimeric species under relaxed folding. conditions. The concentration ofdenaturant in the solution then is decreased in a controlled, preferablystep-wise manner, so as to allow proper refolding of the dimer and proregions while maintaining the association of the pro domain with thedimer. Useful denaturants include 4-6M urea or guanidine hydrochloride(GuHCl), in buffered solutions of pH 4-10, preferably pH 6-8. Thesoluble complex then is formed by controlled dialysis or dilution into asolution having a final denaturant concentration of less than 0.1-2Murea or GuHCl, preferably 1-2 M urea of GuHCl, which then preferably canbe diluted into a physiological buffer. Protein purification/renaturingprocedures and considerations are well described in the art, and detailsfor developing a suitable renaturing protocol readily can be determinedby one having ordinary skill in the art. One useful text one the subjectis Guide to Protein Purification, M. Deutscher, ed., Academic Press, SanDiego, 1990, particularly section V. Complex formation also may be aidedby addition of one or more chaperone proteins.

VI. Stability of Soluble Morphogen Complexes

The stability of the highly purified soluble morphogen complex in aphysiological buffer, e.g., tris-buffered saline (TBS) andphosphate-buffered saline (PBS), can be enhanced by any of a number ofmeans. Currently preferred is by means of a pro region that comprises atleast the first 18 amino acids of the pro sequence (e.g., residues 30-47of Seq. ID NO. 1 for OP-1), and preferably is the full length proregion. Residues 30-47 show sequence homology to the N-terminal portionof other morphogens and are believed to have particular utility inenhancing complex stability for all morphogens. Other useful means forenhancing the stability of soluble morphogen complexes include threeclasses of additive. These additives include basic amino acids (e.g.,L-arginine, lysine and betaine); nonionic detergents (e.g., Tween 80 orNonidet P-120); and carrier proteins (e.g., serum albumin and casein).These additives include 1-100 mM, preferably 10-70 mM, including 50 mM,basic amino acid;, 0.01-1.0%, preferably 0.05-0.2%, including 0.1% (v/v)nonionic detergent;, and 0.01-1.0%, preferably 0.05-0.2%, including 0.1%(w/v) carrier protein.

VII. Activity of Soluble Morphogen Complex

Association of the pro domain with the mature dimeric species does notinterfere with the morphogenic activity of the protein in vivo asdemonstrated by different activity assays. Specifically, soluble OP-1complex provided in a standard rat osteopenia model induces significantincrease in bone growth and osteocalcin production (see Table II,below), in a mannor analogous to the results obtained using maturemorphogen.

The assay is analogous to the osteoporosis model described in U.S. Ser.No. 923,780, now abandoned in favor of continuation application U.S.Ser. No. 08/432,883, but uses aged female rats rather thanovariectomized animals. Briefly, young or aged female rats (CharlesRiver Labs, 115-145, and 335-460 g body weight, respectively) were doseddaily for 7 days by intravenous tail injection, with either 20 μg/Kgbody weight soluble OP-1, or 100 μg/Kg body weight soluble OP-1. Controlgroups of young and aged female rats were dosed only with tris-bufferedsaline (TBS). Water and food were provided to all animals ad libitum.After 14 days, animals were sacrificed, and new bone growth measured bystandard histometric procedures. Osteocalcin concentrations in serumalso were measured. No detrimental effects of morphogen administrationwere detected as determined by changes in animal body or organ weight orby hematology profiles.

                  TABLE II                                                        ______________________________________                                        No.                  Bone Area   Osteocalcin                                  Animals  Animal Group                                                                              (B.Ar/T.Ar) (ng/ml)                                      ______________________________________                                        4        Control     5.50 ± 0.64                                                                            11.89 ± 4.20                              5        Aged female,                                                                              7.68 ± 0.63**                                                                          22.24 ± 2.28**                                     20 μg/Kg                                                                   sol. OP-1                                                            5        Aged female,                                                                              9.82 ± 3.31*                                                                           20.87 ± 6.14*                                      100 μg/Kg                                                                  sol. OP-1                                                            ______________________________________                                         *P < 0.05                                                                     **P < 0.01                                                               

Similar experiments performed using soluble OP-1 complex in theosteoporosis model described in U.S. Ser. No. 923,780, now abandoned infavor of continuation application U.S. Ser. No. 08/432,883 andincorporated hereinabove by reference using ovariectomized rats alsoshow no detrimental effect using the complex form.

Both mature and soluble morphogen also can induce CAM (cell adhesionmolecule) expression, as described in copending U.S. Ser. No.07/922,8133, filed Jul. 31, 1992 now abandoned in favor of continuationapplication U.S. Ser. No. 08/260,675, the disclosure of which isincorporated hereinabove by reference.

Briefly, and as described therein, induction of N-CAM isoforms(N-CAM-180, N-CAM-140 and N-CAM-120) can be monitored by reaction withthe commercially available antibody mAb H28.123 (Sigma Co., St. Louis)and standard Western blot analysis (see, for example, Molecular Cloning,A Laboratory Manual, Sambrook et al. eds. Cold Spring Harbor Press, NewYork, 1989, particularly Section 18). Incubation of a growing culture oftransformed cells of neuronal origin, NG108-15 cels (ATCC, Rockville,Md.) with either mature morphogen dimers or soluble morphogen complexes(10-100 ng/ml, preferably at least 40 ng/ml) induces a redifferentiationof these cells back to a morphology characteristic of untransformedneurons, including specific induction and/or enhanced expression of all3 N-CAM isoforms. In the experiment, cells were subcultured onpoly-L-lysine coated 6-well plates and grown in chemically definedmedium for 2 days before the experiment. Fresh aliquots of morphogenwere added (2.5 μl ) daily.

VIII. Antibody Production

Provided below are standard protocols for polycolonal and monoclonalantibody production. For antibodies which recognize the soluble complexonly, preferably the isolated pro region is used as the antigen; whereantibodies specific to the mature protein are desired, the antigenpreferably comprises at least the C-terminal domain or the intact maturesequence.

Polyclonal antibody may be prepared as follows. Each rabbit is given aprimary immunization of 100 ug/500 μl of antigen, in 0.1% SDS mixed with500 μl Complete Freund's Adjuvant. The antigen is injectedsubcutaneously at multiple sites on the back and flanks of the animal.The rabbit is boosted after a month in the same manner using incompleteFreund's Adjuvant. Test bleeds are taken from the ear vein seven dayslater. Two additional boosts and test bleeds are performed at monthlyintervals until antibody against the morphogen antigen is detected inthe serum using an ELISA assay. Then, the rabbit is boosted monthly with100 μg of antigen and bled (15 ml per bleed) at days seven and ten afterboosting.

Monoclonal antibody specific for a given morphogen may be prepared asfollows. A mouse is given two injections of the morphogen antigen. Theprotein or protein fragment preferably is recombinantly produced. Thefirst injection contains 100 μg of antigen in complete Freund's adjuvantand is given subcutaneously. The second injection contains 50 μg ofantigen in incomplete adjuvant and is given intraperitoneally. The mousethen receives a total of 230 μg of OP-3 in four intraperitonealinjections at various times over an eight month period. One week priorto fusion, the mouse is boosted intraperitoneally with antigen (e.g.,100 μg) and may be additionally boosted with a peptide fragmentconjugated to bovine serum albumin with a suitable crosslinking agent.This boost can be repeated five days (IP), four days (IP), three days(IP) and one day (IV) prior to fusion. The mouse spleen cells then arefused to commercially available myeloma cells at a ratio of 1:1 usingPEG 1500 (Boehringer Mannheim, Germany), and the fused cells plated andscreened for mature or soluble morphogen-specific antibodies using theappropriate portion of the morphogen sequence as antigen. The cellfusion and monoclonal screening steps readily are performed according tostandard procedures well described in standard texts widely available inthe art.

Using these standard procedures, anti-pro domain antisera was preparedfrom rabbits using the isolated pro domain from OP-1 as the antigen, andmonoclonal antibody ("mAb") to the mature domain was produced in mice,using an E. coli-produced truncated form of OP-1 as antigen.

Standard Western blot analysis performed under reducing conditionsdemonstrates that the anti-pro domain antisera ("anti-pro") is specificfor the pro domain only, while the mAb to mature OP-1 ("anti-matureOP-1") is specific for the dimer subunits, that the two antibodies donot cross-react, and that the antibodies and can be used to distinguishbetween soluble and mature protein forms in a sample, e.g., ofconditioned media or serum. A tabular representation of the Western blotresults is in Table III below, where reactivity of mAb to mature OP-1 isindicated by "yy", and reactivity of the anti-pro antisera is indicatedby "xx".

                  TABLE III                                                       ______________________________________                                                                              Purified                                         Purified Conditioned Isolated                                                                              Dimer                                   Antibody Sol OP1  CHO Cell Media                                                                            Pro Domain                                                                            Subunits                                ______________________________________                                        "anti-pro"                                                                             xx       xx          xx                                              "anti-   yy       yy                  yy                                      mature OP-1"                                                                  ______________________________________                                    

IX. Immunoassays

The ability to detect morphogens in solution and to distinguish betweensoluble and mature dimeric morphogen forms provides a valuable tool fordiagnostic assays, allowing one to monitor the level and type ofmorphogen free in the body, e.g., in serum and other body fluids.

For example, OP-1 is an intimate participant in normal bone growth andresorption. Thus, soluble OP-1 is expected to be detected at higherconcentrations in individuals experiencing high bone turnover, such aschildren, and at substantially lower levels in individuals withabnormally low rates of bone turnover, such as patients withosteoporosis, osteosarcoma, Paget's disease and the like. Monitoring thelevel of OP-1, or other bone targeted morphogens such as BMP2 and BMP4,in serum thus provides a means for evaluating the status of bone tissuein an individual, as well as a means for monitoring the efficacy of atreatment to regenerate damaged or lost bone tissue. Similarly,monitoring the level of endogenous GDF-1, can provide diagnosticinformation on the health of nerve tissue, particularly brain tissue.Moreover, following this disclosure one can distinguish between thelevel of soluble and mature forms in solution.

A currently preferred detection means for evaluating the level ofmorphogen in a body fluid comprises an immunoassay utilizing an antibodyor other suitable binding protein capable of reacting specifically witha morphogen and being detected as part of a complex with the morphogen.Immunoassays may be performed using standard techniques known in the artand antibodies raised against a morphogen and specific for thatmorphogen. Antibodies which recognize a morphogen protein form ofinterest may be generated as described herein and these antibodies thenused to monitor endogenous levels of protein in a body fluid, such asserum, whole blood or peritoneal fluid. To monitor endogenousconcentrations of soluble morphogen, the antibody chosen preferably hasbinding specificity for the soluble form e.g., has specificity for thepro domain. Such antibodies may be generated by using the pro domain ora portion thereof as the antigen, essentially as described herein. Asuitable pro domain for use as an antigen may be obtained by isolatingthe soluble complex and then separating the noncovalently associated prodomain from the mature domain using standard procedures, e.g., bypassing the complex over an HPLC column, as described above or byseparation by gel electrophoresis. Alternatively, the pro form of theprotein in its monomeric form may be used as the antigen and thecandidate antibodies screened by Western blot or other standardimmunoassay for those which recognize the pro domain of the soluble formof the protein of interest, but not the mature form, also as describedabove.

Monomeric pro forms can be obtained from cell lysates of CHO producedcells, or from prokaryotic expression of a DNA encoding the pro form, infor example, E. coli. The pro form, which has an apparent molecularweight of about 50 kDa in mammalian cells, can then be isolated by HPLCand/or by gel electrophoresis, as described above.

In order to detect and/or quantitate the amount of morphogenic proteinpresent in a solution, an immunoassay may be performed to detect themorphogen using a polyclonal or monoclonal antibody specific for thatprotein. Here, soluble and mature forms of the morphogen also may bedistinguished by using antibodies that discriminate between the twoforms of the proteins as described above. Currently preferred assaysinclude ELISAS and radioimmunassays, including standard competitorassays useful for quantitating the morphogen in a sample, where anunknown amount of sample morphogen is allowed to react withanti-morphogen antibody and this interaction is competed with a knownamount of labeled antigen. The level of bound or free labeled antigen atequilibrium then is measured to quantitate the amount of unlabeledantigen in solution, the amount of sample antigen being proportional tothe amount of free labeled antigen. Exemplary protocols for these assaysare provided below. However, as will be appreciated by those skilled inthe art, variations of these protocols, as well as other immunoassays,are well known in the literature and within the skill of the art. Forexample, in the ELISA protocol provided below, soluble OP-1 isidentified in a sample using biotinylated anti-pro antiserum.Biotinylated antibodies can be visualized in a colormetric assay or in achemiluminescent assay, as described below. Alternatively, the antibodycan be radio-labeled with a suitable molecule, such as ¹²⁵ I. Stillanother protocol that may be used is a solid phase immunoassay,preferably using an affinity column with anti-morphogen antibodycomplexed to the matrix surface and over which a serum sample may bepassed. A detailed description of useful immunoassays, includingprotocols and general considerations is provided in, for example,Molecular Cloning: A Laboratory Manual, Sambrook et al., eds. ColdSpring Harbor Press, New York, 1989, particularly Section 18.

For serum assays, the serum preferably first is partially purified toremove some of the excess, contaminating serum proteins, such as serumalbumin. Preferably the serum is extracted by precipitation in ammoniumsulfate (e.g., 45%) such that the complex is precipitated. Furtherpurification can be achieved using purification strategies that takeadvantage of the differential solubility of soluble morphogen complex ormature morphogens relative to that of the other proteins present inserum. Further purification also can be achieved by chromatographictechniques well known in the art.

Soluble OP-1 may be detected using a polyclonal antibody specific forthe OP-1 pro domain in an ELISA, as follows. 1 μg/100 μl ofaffinity-purified polyclonal rabbit IgG specific for OP-1-pro is addedto each well of a 96-well plate and incubated at 37° C. for an hour. Thewells are washed four times with 0.167M sodium borate buffer with 0.15 MNaCl (BSB), pH 8.2, containing 0.1% Tween 20. To minimize non-specificbinding, the wells are blocked by filling completely with 1% bovineserum albumin (BSA) in BSB and incubating for 1 hour at 37° C. The wellsare then washed four times with BSB containing 0.1% Tween 20. A 100 μlaliquot of an appropriate dilution of each of the test samples of cellculture supernatant or serum sample is added to each well in triplicateand incubated at 37° C. for 30 min. After incubation, 100 μlbiotinylated rabbit anti-pro serum (stock solution is about 1 mg/ml anddiluted 1:400 in BSB containing 1% BSA before use) is added to each welland incubated at 37° C. for 30 min. The wells are then washed four timeswith BSB containing 0.1% Tween 20. 100 μl strepavidin-alkaline (SouthernBiotechnology Associates, Inc. Birmingham, Ala., diluted 1:2000 in BSBcontaining 0.1% Tween 20 before use) is added to each well and incubatedat 37° C. for 30 min. The plates are washed four times with 0.5M Trisbuffered Saline (TBS), pH 7.2. 50 μl substrate (ELISA AmplificationSystem Kit, Life Technologies, Inc., Bethesda, Md.) is added to eachwell incubated at room temperature for 15 min. Then, 50 μl amplifier(from the same amplification system kit) is added and incubated foranother 15 min at room temperature. The reaction is stopped by theaddition of 50 μl 0.3 M sulphuric acid. The OD at 490 nm of the solutionin each well is recorded. To quantitate the level of soluble OP-1 in thesample, a standard curve is performed in parallel with the test samples.In the standard curve, known increasing amounts of purified OP-1-pro isadded. Alternatively, using, for example, Lumi-phos 530 (AnalyticalLuminescence Laboratories) as the substrate and detection at 300-650 nmin a standard luminometer, complexes can be detected bychemiluminescence, which typically provides a more sensitive assay thandetection by means of a visible color change.

Morphogen (soluble or mature form) may be detected in a standardplated-based radioimmunoassay as follows. Empirically determinedlimiting levels of anti-morphogen antibody (e.g., anti-OP-1, typically50-80 ng/well) are bound to wells of a PVC plate e.g., in 50 μl PBSphosphate buffered saline. After sufficient incubation to allow bindingat room temperature, typically one hour, the plate is washed in aPBS/Tween 20 solution, ("washing buffer"), and 200 μl of block (3% BSA,0.1 μ lysine in 1×BSB) is added to each well and allowed to incubate for1 hour, after which the wells are washed again in washing buffer. 40 μlof a sample composed of serially diluted plasma (preferably partiallypurified as described above) or morphogen standard (e.g., OP-1) is addedto wells in triplicate. Samples preferably are diluted in PTTH (15 mMKH₂ PO₄, 8 mM Na₂ PO₄, 27 mM KCl, 137 mM NaCl, 0.05% Tween 20, 1 mg/mlHSA, 0.05% NaN₃, pH 7.2). 10 μl of labelled competitor antigen,preferably 100,000-500,000 cpm/sample is added (e.g., ¹²⁵ I OP-1,radiolabelled using standard procedures), and plates are incubatedovernight at 4° C. Plates then are washed in washing buffer, and allowedto dry. Wells are cut apart and bound labelled OP-1 counted in astandard gamma counter. The quantities of bound labelled antigen (e.g.,¹²⁵ I OP-1) measured in the presence and absence of sample then arecompared, the difference being proportional to the amount of sampleantigen (morphogen) present in the sample fluid.

As a corollary assay method, immunoassays may be developed to detectendogenous anti-morphogen antibodies, and to distinguish between suchantibodies to soluble or mature forms. Endogenous anti-morphogenantibodies have been detected in serum, and their level is known toincrease, for example, upon implanting of an osteogenic device in amammal. Without being limited to a particular theory, these antibodiesmay play a role in modulating morphogen activity by modulating the levelof available protein in serum. Assays that monitor the level ofendogenous antibodies in blood or their body fluids thus can be used indiagnostic assays to evaluate the status of a tissue, as well as toprovide a means for monitoring the efficacy of a therapy for tissueregeneration.

The currently preferred means for detecting endogenous anti-morphogenantibodies is by means of a standard Western blot. See, for example,Molecular Cloning: A Laboratory Manual Sambrook et al., eds., ColdSpring Harbor Press, New York, 1989, particularly pages 18.60-18.75,incorporated herein by reference, for a detailed description of theseassays. Purified mature or soluble morphogen is electrophoresed on anSDS polyacrylamide gel under oxidized or reduced conditions designed toseparate the proteins in solution, and the proteins then transferred toa polyvinylidene difluoride microporus membrane (0.45 μm pore sizes)using standard buffers and procedures. The filter then is incubated withthe serum being tested (at various dilutions). Antibodies bound toeither the pro domain or the mature morphogen domain are detected bymeans of an anti-human antibody protein, e.g., goat anti-human Ig.Titers of the antimorphogen antibodies can be determined by furtherdilution of the serum until no signal is detected.

X. Formulations and Methods for Administering Soluble Morphogens asTherapeutic Agents

The soluble morphogens of this invention are particularly useful astherapeutic agents to regenerate diseased or damaged tissue in a mammal,particularly a human.

The soluble morphogen complexes may be used to particular advantage inregeneration of damaged or diseased lung, heart, liver, kidney, nerve orpancreas tissue, as well as in the transplantation and/or grafting ofthese tissues and bone marrow, skin, gastrointestinal mucosa, and otherliving tissues.

The soluble morphogen complexes described herein may be provided to anindividual by any suitable means, preferably directly or systemically,e.g., parenterally or orally. Where the morphogen is to be provideddirectly (e.g., locally, as by injection, to a desired tissue site), orparenterally, such as by intravenous, subcutaneous, intramuscular,intraorbital, ophthalmic, intraventricular, intracranial, intracapsular,intraspinal, intracisternal, intraperitoneal, buccal, rectal, vaginal,intranasal or by aerosol administration, the soluble morphogen complexpreferably comprises part of an aqueous solution. The solution isphysiologically acceptable so that in addition to delivery of thedesired morphogen to the patient, the solution does not otherwiseadversely affect the patient's electrolyte and volume balance. Theaqueous medium for the soluble morphogen thus may comprise normalphysiologic saline (0.9% NaCl, 0.15M), pH 7-7.4.

Soluble morphogens of this invention are readily purified from culturedcell media into a physiological buffer, as described above. In addition,and as described above, if desired, the soluble complexes may beformulated with one or more additional additives, including basic aminoacids (e.g., L-arginine, lysine, betaine); non-ionic detergents (e.g.Tween-80 or Nonidet-120) and carrier proteins (e.g., serum albumin andcasein).

Useful solutions for oral or parenteral administration may be preparedby any of the methods well known in the pharmaceutical art, described,for example, in Remington's Pharmaceutical Sciences, (Gennaro, A., ed.),Mack Pub., 1990. Formulations may include, for example, polyalkyleneglycols such as polyethylene glycol, oils of vegetable origin,hydrogenated naphthalenes, and the like. Formulations for directadministration, in particular, may include glycerol and othercompositions of high viscosity. Biocompatible, preferably bioresorbablepolymers, including, for example, hyaluronic acid, collagen, tricalciumphosphate, polybutyrate, polylactide, polyglycolide andlactide/glycolide copolymers, may be useful excipients to control therelease of the soluble morphogen in vivo.

Other potentially useful parenteral delivery systems for thesemorphogens include ethylene-vinyl acetate copolymer particles, osmoticpumps, implantable infusion systems, and liposomes. Formulations forinhalation administration may contain as excipients, for example,lactose, or may be aqueous solutions containing, for example,polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or oilysolutions for administration in the form of nasal drops, or as a gel tobe applied intranasally.

The soluble morphogens described herein also may be administered orally.Oral administration of proteins as therapeutics generally is notpracticed as most proteins readily are degraded by digestive enzymes andacids in the mammalian digestive system before they can be absorbed intothe bloodstream. However, the mature domains of the morphogens describedherein typically are acid-stable and protease-resistant (see, forexample, U.S. Pat. No. 4,968,590.) In addition, at least one morphogen,OP-1, has been identified, in mammary gland extract, colostrum and milk,as well as saliva. Moreover, the OP-1 purified from mammary glandextract is morphogenically active. For example, this protein inducesendochondral bone formation in mammals when implanted subcutaneously inassociation with a suitable matrix material, using a standard in vivobone assay, such as is disclosed in U.S. Pat. No. 4,968,590. Inaddition, endogenous morphogen also is detected in human serum (seeabove). Finally, comparative experiments with soluble and maturemorphogens in a number of experiments defining morphogenic activityindicate that the non-covalent association of the pro domain with thedimeric species does not interfere with morphogenic activity. Thesefindings indicate that oral and parenteral administration are viablemeans for administering morphogens to an individual, and that solublemorphogens have utility in systemic administration protocols.

The soluble complexes provided herein also may be associated withmolecules capable of targeting the morphogen to a desired tissue. Forexample, tetracycline and diphosphonates (bisphosphonates) are known tobind to bone mineral, particularly at zones of bone remodeling, whenthey are provided systemically in a mammal. Accordingly, these moleculesmay be included as useful agents for targeting soluble morphogens tobone tissue. Alternatively, an antibody or other binding protein thatinteracts specifically with a surface molecule on the desired targettissue cells also may be used. Such targeting molecules further may becovalently associated to the morphogen complex, e.g., by chemicalcrosslinking, or by using standard genetic engineering means to create,for example, an acid labile bond such as an Asp-Pro linkage. Usefultargeting molecules may be designed, for example, using the single chainbinding site technology disclosed, for example, in U.S. Pat. No.5,091,513.

Finally, the soluble morphogen complexes provided herein may beadministered alone or in combination with other molecules known to havea beneficial effect on tissue morphogenesis, including molecules capableof tissue repair and regeneration and/or inhibiting inflammation.Examples of useful cofactors for stimulating bone tissue growth inosteoporotic individuals, for example, include but are not limited to,vitamin D₃, calcitonin, prostaglandins, parathyroid hormone,dexamethasone, estrogen and IGF-I or IGF-II. Useful cofactors for nervetissue repair and regeneration may include nerve growth factors. Otheruseful cofactors include symptom-alleviating cofactors, includingantiseptics, antibiotics, antiviral and antifungal agents and analgesicsand anesthetics.

The compounds provided herein can be formulated into pharmaceuticalcompositions by admixture with pharmaceutically acceptable nontoxicexcipients and carriers. As noted above, such compositions may beprepared for parenteral administration, particularly in the form ofliquid solutions or suspensions; for oral administration, particularlyin the form of tablets or capsules; or intranasally, particularly in theform of powders, nasal drops or aerosols. Where adhesion to a tissuesurface is desired the composition may include the morphogen dispersedin a fibrinogen-thrombin composition or other bioadhesive such as isdisclosed, for example in PCT US91/09275, the disclosure of which isincorporated herein by reference. The composition then may be painted,sprayed or otherwise applied to the desired tissue surface.

The compositions can be formulated for parenteral or oral administrationto humans or other mammals in therapeutically effective amounts, e.g.,amounts which provide appropriate concentrations of the morphogen totarget tissue for a time sufficient to induce morphogenesis, includingparticular steps thereof, as described above.

Where the soluble morphogen complex is to be used as part of atransplant procedure, the morphogen may be provided to the living tissueor organ to be transplanted prior to removal of the tissue or organ fromthe donor. The morphogen may be provided to the donor host directly, asby injection of a formulation comprising the soluble complex into thetissue, or indirectly, e.g., by oral or parenteral administration, usingany of the means described above.

Alternatively or, in addition, once removed from the donor, the organ orliving tissue may be placed in a preservation solution containing themorphogen. In addition, the recipient also preferably is provided withthe morphogen just prior to, or concommitant with, transplantation. Inall cases, the soluble complex may be administered directly to thetissue at risk, as by injection to the tissue, or it may be providedsystemically, either by oral or parenteral administration, using any ofthe methods and formulations described herein and/or known in the art.

Where the morphogen comprises part of a tissue or organ preservationsolution, any commercially available preservation solution may be usedto advantage. A useful preservation solution is described in U.S. Ser.No. 07/938,337 (WO93/04692), filed Aug. 28, 1992, and in PCT/US92/07358,both incorporated herein by reference.

As will be appreciated by those skilled in the art, the concentration ofthe compounds described in a therapeutic composition will vary dependingupon a number of factors, including the dosage of the drug to beadministered, the chemical characteristics (e.g., hydrophobicity) of thecompounds employed, and the route of administration. The preferreddosage of drug to be administered also is likely to depend on suchvariables as the type and extent of tissue loss or defect, the overallhealth status of the particular patient, the relative biologicalefficacy of the compound selected, the formulation of the compound, thepresence and types of excipients in the formulation, and the route ofadministration. In general terms, the compounds of this invention may beprovided in an aqueous physiological buffer solution containing about0.001 to 10% w/v compound for parenteral administration. Typical doseranges are from about 10 ng/kg to about 1 g/kg of body weight per day; apreferred dose range is from about 0.1 μg/kg to 100 mg/kg of bodyweight. No obvious morphogen-induced pathological lesions are inducedwhen mature morphogen (e.g., OP-1, 20 μg) is administered daily tonormal growing rats for 21 consecutive days. Moreover, 10 μg systemicinjections of morphogen (e.g., OP-1) injected daily for 10 days intonormal newborn mice does not produce any gross abnormalities.

Where morphogens are administered systemically, in the methods of thepresent invention, preferably a large volume loading dose is used at thestart of the treatment. The treatment then is continued with amaintenance dose. Further administration then can be determined bymonitoring at intervals the levels of the morphogen in the blood.

OTHER EMBODIMENTS

The invention may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The presentembodiments are therefore to be considered in all respects asillustrative and not restrictive, the scope of the invention beingindicated by the appended claims rather than by the foregoingdescription, and all changes which come within the meaning and range ofequivalency of the claims are therefore intended to be embraced therein.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES: 23                                            - (2) INFORMATION FOR SEQ ID NO:1:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 1822 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: HOMO SAPI - #ENS                                                (F) TISSUE TYPE: HIPPOC - #AMPUS                                    -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 49..1341                                                        (C) IDENTIFICATION METHOD: - # experimental                         #/function= "OSTEOGENIC PROTEIN"                                                             /product=- # "OP1"                                                            /evidence=- # EXPERIMENTAL                                                    /standard.sub.-- - #name= "OP1"                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                 #CAC GTG      57AGCCCGG AGCCCGGGTA GCGCGTAGAG CCGGCGCG ATG                    #                 Met - # His Val                                             # 1                                                                           - CGC TCA CTG CGA GCT GCG GCG CCG CAC AGC TT - #C GTG GCG CTC TGG GCA          105                                                                          Arg Ser Leu Arg Ala Ala Ala Pro His Ser Ph - #e Val Ala Leu Trp Ala           #      15                                                                     - CCC CTG TTC CTG CTG CGC TCC GCC CTG GCC GA - #C TTC AGC CTG GAC AAC          153                                                                          Pro Leu Phe Leu Leu Arg Ser Ala Leu Ala As - #p Phe Ser Leu Asp Asn           # 35                                                                          - GAG GTG CAC TCG AGC TTC ATC CAC CGG CGC CT - #C CGC AGC CAG GAG CGG          201                                                                          Glu Val His Ser Ser Phe Ile His Arg Arg Le - #u Arg Ser Gln Glu Arg           #                 50                                                          - CGG GAG ATG CAG CGC GAG ATC CTC TCC ATT TT - #G GGC TTG CCC CAC CGC          249                                                                          Arg Glu Met Gln Arg Glu Ile Leu Ser Ile Le - #u Gly Leu Pro His Arg           #             65                                                              - CCG CGC CCG CAC CTC CAG GGC AAG CAC AAC TC - #G GCA CCC ATG TTC ATG          297                                                                          Pro Arg Pro His Leu Gln Gly Lys His Asn Se - #r Ala Pro Met Phe Met           #         80                                                                  - CTG GAC CTG TAC AAC GCC ATG GCG GTG GAG GA - #G GGC GGC GGG CCC GGC          345                                                                          Leu Asp Leu Tyr Asn Ala Met Ala Val Glu Gl - #u Gly Gly Gly Pro Gly           #     95                                                                      - GGC CAG GGC TTC TCC TAC CCC TAC AAG GCC GT - #C TTC AGT ACC CAG GGC          393                                                                          Gly Gln Gly Phe Ser Tyr Pro Tyr Lys Ala Va - #l Phe Ser Thr Gln Gly           100                 1 - #05                 1 - #10                 1 -       #15                                                                           - CCC CCT CTG GCC AGC CTG CAA GAT AGC CAT TT - #C CTC ACC GAC GCC GAC          441                                                                          Pro Pro Leu Ala Ser Leu Gln Asp Ser His Ph - #e Leu Thr Asp Ala Asp           #               130                                                           - ATG GTC ATG AGC TTC GTC AAC CTC GTG GAA CA - #T GAC AAG GAA TTC TTC          489                                                                          Met Val Met Ser Phe Val Asn Leu Val Glu Hi - #s Asp Lys Glu Phe Phe           #           145                                                               - CAC CCA CGC TAC CAC CAT CGA GAG TTC CGG TT - #T GAT CTT TCC AAG ATC          537                                                                          His Pro Arg Tyr His His Arg Glu Phe Arg Ph - #e Asp Leu Ser Lys Ile           #       160                                                                   - CCA GAA GGG GAA GCT GTC ACG GCA GCC GAA TT - #C CGG ATC TAC AAG GAC          585                                                                          Pro Glu Gly Glu Ala Val Thr Ala Ala Glu Ph - #e Arg Ile Tyr Lys Asp           #   175                                                                       - TAC ATC CGG GAA CGC TTC GAC AAT GAG ACG TT - #C CGG ATC AGC GTT TAT          633                                                                          Tyr Ile Arg Glu Arg Phe Asp Asn Glu Thr Ph - #e Arg Ile Ser Val Tyr           180                 1 - #85                 1 - #90                 1 -       #95                                                                           - CAG GTG CTC CAG GAG CAC TTG GGC AGG GAA TC - #G GAT CTC TTC CTG CTC          681                                                                          Gln Val Leu Gln Glu His Leu Gly Arg Glu Se - #r Asp Leu Phe Leu Leu           #               210                                                           - GAC AGC CGT ACC CTC TGG GCC TCG GAG GAG GG - #C TGG CTG GTG TTT GAC          729                                                                          Asp Ser Arg Thr Leu Trp Ala Ser Glu Glu Gl - #y Trp Leu Val Phe Asp           #           225                                                               - ATC ACA GCC ACC AGC AAC CAC TGG GTG GTC AA - #T CCG CGG CAC AAC CTG          777                                                                          Ile Thr Ala Thr Ser Asn His Trp Val Val As - #n Pro Arg His Asn Leu           #       240                                                                   - GGC CTG CAG CTC TCG GTG GAG ACG CTG GAT GG - #G CAG AGC ATC AAC CCC          825                                                                          Gly Leu Gln Leu Ser Val Glu Thr Leu Asp Gl - #y Gln Ser Ile Asn Pro           #   255                                                                       - AAG TTG GCG GGC CTG ATT GGG CGG CAC GGG CC - #C CAG AAC AAG CAG CCC          873                                                                          Lys Leu Ala Gly Leu Ile Gly Arg His Gly Pr - #o Gln Asn Lys Gln Pro           260                 2 - #65                 2 - #70                 2 -       #75                                                                           - TTC ATG GTG GCT TTC TTC AAG GCC ACG GAG GT - #C CAC TTC CGC AGC ATC          921                                                                          Phe Met Val Ala Phe Phe Lys Ala Thr Glu Va - #l His Phe Arg Ser Ile           #               290                                                           - CGG TCC ACG GGG AGC AAA CAG CGC AGC CAG AA - #C CGC TCC AAG ACG CCC          969                                                                          Arg Ser Thr Gly Ser Lys Gln Arg Ser Gln As - #n Arg Ser Lys Thr Pro           #           305                                                               - AAG AAC CAG GAA GCC CTG CGG ATG GCC AAC GT - #G GCA GAG AAC AGC AGC         1017                                                                          Lys Asn Gln Glu Ala Leu Arg Met Ala Asn Va - #l Ala Glu Asn Ser Ser           #       320                                                                   - AGC GAC CAG AGG CAG GCC TGT AAG AAG CAC GA - #G CTG TAT GTC AGC TTC         1065                                                                          Ser Asp Gln Arg Gln Ala Cys Lys Lys His Gl - #u Leu Tyr Val Ser Phe           #   335                                                                       - CGA GAC CTG GGC TGG CAG GAC TGG ATC ATC GC - #G CCT GAA GGC TAC GCC         1113                                                                          Arg Asp Leu Gly Trp Gln Asp Trp Ile Ile Al - #a Pro Glu Gly Tyr Ala           340                 3 - #45                 3 - #50                 3 -       #55                                                                           - GCC TAC TAC TGT GAG GGG GAG TGT GCC TTC CC - #T CTG AAC TCC TAC ATG         1161                                                                          Ala Tyr Tyr Cys Glu Gly Glu Cys Ala Phe Pr - #o Leu Asn Ser Tyr Met           #               370                                                           - AAC GCC ACC AAC CAC GCC ATC GTG CAG ACG CT - #G GTC CAC TTC ATC AAC         1209                                                                          Asn Ala Thr Asn His Ala Ile Val Gln Thr Le - #u Val His Phe Ile Asn           #           385                                                               - CCG GAA ACG GTG CCC AAG CCC TGC TGT GCG CC - #C ACG CAG CTC AAT GCC         1257                                                                          Pro Glu Thr Val Pro Lys Pro Cys Cys Ala Pr - #o Thr Gln Leu Asn Ala           #       400                                                                   - ATC TCC GTC CTC TAC TTC GAT GAC AGC TCC AA - #C GTC ATC CTG AAG AAA         1305                                                                          Ile Ser Val Leu Tyr Phe Asp Asp Ser Ser As - #n Val Ile Leu Lys Lys           #   415                                                                       - TAC AGA AAC ATG GTG GTC CGG GCC TGT GGC TG - #C CAC TAGCTCCTCC              1351                                                                          Tyr Arg Asn Met Val Val Arg Ala Cys Gly Cy - #s His                           420                 4 - #25                 4 - #30                           - GAGAATTCAG ACCCTTTGGG GCCAAGTTTT TCTGGATCCT CCATTGCTCG CC - #TTGGCCAG       1411                                                                          - GAACCAGCAG ACCAACTGCC TTTTGTGAGA CCTTCCCCTC CCTATCCCCA AC - #TTTAAAGG       1471                                                                          - TGTGAGAGTA TTAGGAAACA TGAGCAGCAT ATGGCTTTTG ATCAGTTTTT CA - #GTGGCAGC       1531                                                                          - ATCCAATGAA CAAGATCCTA CAAGCTGTGC AGGCAAAACC TAGCAGGAAA AA - #AAAACAAC       1591                                                                          - GCATAAAGAA AAATGGCCGG GCCAGGTCAT TGGCTGGGAA GTCTCAGCCA TG - #CACGGACT       1651                                                                          - CGTTTCCAGA GGTAATTATG AGCGCCTACC AGCCAGGCCA CCCAGCCGTG GG - #AGGAAGGG       1711                                                                          - GGCGTGGCAA GGGGTGGGCA CATTGGTGTC TGTGCGAAAG GAAAATTGAC CC - #GGAAGTTC       1771                                                                          #           1822CACAATA AAACGAATGA ATGAAAAAAA AAAAAAAAAA A                    - (2) INFORMATION FOR SEQ ID NO:2:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 431 amino                                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                 - Met His Val Arg Ser Leu Arg Ala Ala Ala Pr - #o His Ser Phe Val Ala         #                 15                                                          - Leu Trp Ala Pro Leu Phe Leu Leu Arg Ser Al - #a Leu Ala Asp Phe Ser         #             30                                                              - Leu Asp Asn Glu Val His Ser Ser Phe Ile Hi - #s Arg Arg Leu Arg Ser         #         45                                                                  - Gln Glu Arg Arg Glu Met Gln Arg Glu Ile Le - #u Ser Ile Leu Gly Leu         #     60                                                                      - Pro His Arg Pro Arg Pro His Leu Gln Gly Ly - #s His Asn Ser Ala Pro         # 80                                                                          - Met Phe Met Leu Asp Leu Tyr Asn Ala Met Al - #a Val Glu Glu Gly Gly         #                 95                                                          - Gly Pro Gly Gly Gln Gly Phe Ser Tyr Pro Ty - #r Lys Ala Val Phe Ser         #           110                                                               - Thr Gln Gly Pro Pro Leu Ala Ser Leu Gln As - #p Ser His Phe Leu Thr         #       125                                                                   - Asp Ala Asp Met Val Met Ser Phe Val Asn Le - #u Val Glu His Asp Lys         #   140                                                                       - Glu Phe Phe His Pro Arg Tyr His His Arg Gl - #u Phe Arg Phe Asp Leu         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Ser Lys Ile Pro Glu Gly Glu Ala Val Thr Al - #a Ala Glu Phe Arg Ile         #               175                                                           - Tyr Lys Asp Tyr Ile Arg Glu Arg Phe Asp As - #n Glu Thr Phe Arg Ile         #           190                                                               - Ser Val Tyr Gln Val Leu Gln Glu His Leu Gl - #y Arg Glu Ser Asp Leu         #       205                                                                   - Phe Leu Leu Asp Ser Arg Thr Leu Trp Ala Se - #r Glu Glu Gly Trp Leu         #   220                                                                       - Val Phe Asp Ile Thr Ala Thr Ser Asn His Tr - #p Val Val Asn Pro Arg         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - His Asn Leu Gly Leu Gln Leu Ser Val Glu Th - #r Leu Asp Gly Gln Ser         #               255                                                           - Ile Asn Pro Lys Leu Ala Gly Leu Ile Gly Ar - #g His Gly Pro Gln Asn         #           270                                                               - Lys Gln Pro Phe Met Val Ala Phe Phe Lys Al - #a Thr Glu Val His Phe         #       285                                                                   - Arg Ser Ile Arg Ser Thr Gly Ser Lys Gln Ar - #g Ser Gln Asn Arg Ser         #   300                                                                       - Lys Thr Pro Lys Asn Gln Glu Ala Leu Arg Me - #t Ala Asn Val Ala Glu         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Asn Ser Ser Ser Asp Gln Arg Gln Ala Cys Ly - #s Lys His Glu Leu Tyr         #               335                                                           - Val Ser Phe Arg Asp Leu Gly Trp Gln Asp Tr - #p Ile Ile Ala Pro Glu         #           350                                                               - Gly Tyr Ala Ala Tyr Tyr Cys Glu Gly Glu Cy - #s Ala Phe Pro Leu Asn         #       365                                                                   - Ser Tyr Met Asn Ala Thr Asn His Ala Ile Va - #l Gln Thr Leu Val His         #   380                                                                       - Phe Ile Asn Pro Glu Thr Val Pro Lys Pro Cy - #s Cys Ala Pro Thr Gln         385                 3 - #90                 3 - #95                 4 -       #00                                                                           - Leu Asn Ala Ile Ser Val Leu Tyr Phe Asp As - #p Ser Ser Asn Val Ile         #               415                                                           - Leu Lys Lys Tyr Arg Asn Met Val Val Arg Al - #a Cys Gly Cys His             #           430                                                               - (2) INFORMATION FOR SEQ ID NO:3:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 1873 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 104..1393                                             #/function= "OSTEOGENIC PROTEIN"                                                             /product=- # "MOP1"                                            #"MOP1 CDNA"   /note=                                                         -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                 - CTGCAGCAAG TGACCTCGGG TCGTGGACCG CTGCCCTGCC CCCTCCGCTG CC - #ACCTGGGG         60                                                                          - CGGCGCGGGC CCGGTGCCCC GGATCGCGCG TAGAGCCGGC GCG ATG CA - #C GTG CGC          115                                                                          #            Met His Val Ar - #g                                              #              1                                                              - TCG CTG CGC GCT GCG GCG CCA CAC AGC TTC GT - #G GCG CTC TGG GCG CCT          163                                                                          Ser Leu Arg Ala Ala Ala Pro His Ser Phe Va - #l Ala Leu Trp Ala Pro           #  20                                                                         - CTG TTC TTG CTG CGC TCC GCC CTG GCC GAT TT - #C AGC CTG GAC AAC GAG          211                                                                          Leu Phe Leu Leu Arg Ser Ala Leu Ala Asp Ph - #e Ser Leu Asp Asn Glu           #                 35                                                          - GTG CAC TCC AGC TTC ATC CAC CGG CGC CTC CG - #C AGC CAG GAG CGG CGG          259                                                                          Val His Ser Ser Phe Ile His Arg Arg Leu Ar - #g Ser Gln Glu Arg Arg           #             50                                                              - GAG ATG CAG CGG GAG ATC CTG TCC ATC TTA GG - #G TTG CCC CAT CGC CCG          307                                                                          Glu Met Gln Arg Glu Ile Leu Ser Ile Leu Gl - #y Leu Pro His Arg Pro           #         65                                                                  - CGC CCG CAC CTC CAG GGA AAG CAT AAT TCG GC - #G CCC ATG TTC ATG TTG          355                                                                          Arg Pro His Leu Gln Gly Lys His Asn Ser Al - #a Pro Met Phe Met Leu           #     80                                                                      - GAC CTG TAC AAC GCC ATG GCG GTG GAG GAG AG - #C GGG CCG GAC GGA CAG          403                                                                          Asp Leu Tyr Asn Ala Met Ala Val Glu Glu Se - #r Gly Pro Asp Gly Gln           #100                                                                          - GGC TTC TCC TAC CCC TAC AAG GCC GTC TTC AG - #T ACC CAG GGC CCC CCT          451                                                                          Gly Phe Ser Tyr Pro Tyr Lys Ala Val Phe Se - #r Thr Gln Gly Pro Pro           #               115                                                           - TTA GCC AGC CTG CAG GAC AGC CAT TTC CTC AC - #T GAC GCC GAC ATG GTC          499                                                                          Leu Ala Ser Leu Gln Asp Ser His Phe Leu Th - #r Asp Ala Asp Met Val           #           130                                                               - ATG AGC TTC GTC AAC CTA GTG GAA CAT GAC AA - #A GAA TTC TTC CAC CCT          547                                                                          Met Ser Phe Val Asn Leu Val Glu His Asp Ly - #s Glu Phe Phe His Pro           #       145                                                                   - CGA TAC CAC CAT CGG GAG TTC CGG TTT GAT CT - #T TCC AAG ATC CCC GAG          595                                                                          Arg Tyr His His Arg Glu Phe Arg Phe Asp Le - #u Ser Lys Ile Pro Glu           #   160                                                                       - GGC GAA CGG GTG ACC GCA GCC GAA TTC AGG AT - #C TAT AAG GAC TAC ATC          643                                                                          Gly Glu Arg Val Thr Ala Ala Glu Phe Arg Il - #e Tyr Lys Asp Tyr Ile           165                 1 - #70                 1 - #75                 1 -       #80                                                                           - CGG GAG CGA TTT GAC AAC GAG ACC TTC CAG AT - #C ACA GTC TAT CAG GTG          691                                                                          Arg Glu Arg Phe Asp Asn Glu Thr Phe Gln Il - #e Thr Val Tyr Gln Val           #               195                                                           - CTC CAG GAG CAC TCA GGC AGG GAG TCG GAC CT - #C TTC TTG CTG GAC AGC          739                                                                          Leu Gln Glu His Ser Gly Arg Glu Ser Asp Le - #u Phe Leu Leu Asp Ser           #           210                                                               - CGC ACC ATC TGG GCT TCT GAG GAG GGC TGG TT - #G GTG TTT GAT ATC ACA          787                                                                          Arg Thr Ile Trp Ala Ser Glu Glu Gly Trp Le - #u Val Phe Asp Ile Thr           #       225                                                                   - GCC ACC AGC AAC CAC TGG GTG GTC AAC CCT CG - #G CAC AAC CTG GGC TTA          835                                                                          Ala Thr Ser Asn His Trp Val Val Asn Pro Ar - #g His Asn Leu Gly Leu           #   240                                                                       - CAG CTC TCT GTG GAG ACC CTG GAT GGG CAG AG - #C ATC AAC CCC AAG TTG          883                                                                          Gln Leu Ser Val Glu Thr Leu Asp Gly Gln Se - #r Ile Asn Pro Lys Leu           245                 2 - #50                 2 - #55                 2 -       #60                                                                           - GCA GGC CTG ATT GGA CGG CAT GGA CCC CAG AA - #C AAG CAA CCC TTC ATG          931                                                                          Ala Gly Leu Ile Gly Arg His Gly Pro Gln As - #n Lys Gln Pro Phe Met           #               275                                                           - GTG GCC TTC TTC AAG GCC ACG GAA GTC CAT CT - #C CGT AGT ATC CGG TCC          979                                                                          Val Ala Phe Phe Lys Ala Thr Glu Val His Le - #u Arg Ser Ile Arg Ser           #           290                                                               - ACG GGG GGC AAG CAG CGC AGC CAG AAT CGC TC - #C AAG ACG CCA AAG AAC         1027                                                                          Thr Gly Gly Lys Gln Arg Ser Gln Asn Arg Se - #r Lys Thr Pro Lys Asn           #       305                                                                   - CAA GAG GCC CTG AGG ATG GCC AGT GTG GCA GA - #A AAC AGC AGC AGT GAC         1075                                                                          Gln Glu Ala Leu Arg Met Ala Ser Val Ala Gl - #u Asn Ser Ser Ser Asp           #   320                                                                       - CAG AGG CAG GCC TGC AAG AAA CAT GAG CTG TA - #C GTC AGC TTC CGA GAC         1123                                                                          Gln Arg Gln Ala Cys Lys Lys His Glu Leu Ty - #r Val Ser Phe Arg Asp           325                 3 - #30                 3 - #35                 3 -       #40                                                                           - CTT GGC TGG CAG GAC TGG ATC ATT GCA CCT GA - #A GGC TAT GCT GCC TAC         1171                                                                          Leu Gly Trp Gln Asp Trp Ile Ile Ala Pro Gl - #u Gly Tyr Ala Ala Tyr           #               355                                                           - TAC TGT GAG GGA GAG TGC GCC TTC CCT CTG AA - #C TCC TAC ATG AAC GCC         1219                                                                          Tyr Cys Glu Gly Glu Cys Ala Phe Pro Leu As - #n Ser Tyr Met Asn Ala           #           370                                                               - ACC AAC CAC GCC ATC GTC CAG ACA CTG GTT CA - #C TTC ATC AAC CCA GAC         1267                                                                          Thr Asn His Ala Ile Val Gln Thr Leu Val Hi - #s Phe Ile Asn Pro Asp           #       385                                                                   - ACA GTA CCC AAG CCC TGC TGT GCG CCC ACC CA - #G CTC AAC GCC ATC TCT         1315                                                                          Thr Val Pro Lys Pro Cys Cys Ala Pro Thr Gl - #n Leu Asn Ala Ile Ser           #   400                                                                       - GTC CTC TAC TTC GAC GAC AGC TCT AAT GTC AT - #C CTG AAG AAG TAC AGA         1363                                                                          Val Leu Tyr Phe Asp Asp Ser Ser Asn Val Il - #e Leu Lys Lys Tyr Arg           405                 4 - #10                 4 - #15                 4 -       #20                                                                           - AAC ATG GTG GTC CGG GCC TGT GGC TGC CAC TA - #GCTCTTCC TGAGACCCTG           1413                                                                          Asn Met Val Val Arg Ala Cys Gly Cys His                                       #               430                                                           - ACCTTTGCGG GGCCACACCT TTCCAAATCT TCGATGTCTC ACCATCTAAG TC - #TCTCACTG       1473                                                                          - CCCACCTTGG CGAGGAGAAC AGACCAACCT CTCCTGAGCC TTCCCTCACC TC - #CCAACCGG       1533                                                                          - AAGCATGTAA GGGTTCCAGA AACCTGAGCG TGCAGCAGCT GATGAGCGCC CT - #TTCCTTCT       1593                                                                          - GGCACGTGAC GGACAAGATC CTACCAGCTA CCACAGCAAA CGCCTAAGAG CA - #GGAAAAAT       1653                                                                          - GTCTGCCAGG AAAGTGTCCA GTGTCCACAT GGCCCCTGGC GCTCTGAGTC TT - #TGAGGAGT       1713                                                                          - AATCGCAAGC CTCGTTCAGC TGCAGCAGAA GGAAGGGCTT AGCCAGGGTG GG - #CGCTGGCG       1773                                                                          - TCTGTGTTGA AGGGAAACCA AGCAGAAGCC ACTGTAATGA TATGTCACAA TA - #AAACCCAT       1833                                                                          #  1873            AAAA AAAAAAAAAA AAAAGAATTC                                 - (2) INFORMATION FOR SEQ ID NO:4:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 430 amino                                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                 - Met His Val Arg Ser Leu Arg Ala Ala Ala Pr - #o His Ser Phe Val Ala         #                 15                                                          - Leu Trp Ala Pro Leu Phe Leu Leu Arg Ser Al - #a Leu Ala Asp Phe Ser         #             30                                                              - Leu Asp Asn Glu Val His Ser Ser Phe Ile Hi - #s Arg Arg Leu Arg Ser         #         45                                                                  - Gln Glu Arg Arg Glu Met Gln Arg Glu Ile Le - #u Ser Ile Leu Gly Leu         #     60                                                                      - Pro His Arg Pro Arg Pro His Leu Gln Gly Ly - #s His Asn Ser Ala Pro         # 80                                                                          - Met Phe Met Leu Asp Leu Tyr Asn Ala Met Al - #a Val Glu Glu Ser Gly         #                 95                                                          - Pro Asp Gly Gln Gly Phe Ser Tyr Pro Tyr Ly - #s Ala Val Phe Ser Thr         #           110                                                               - Gln Gly Pro Pro Leu Ala Ser Leu Gln Asp Se - #r His Phe Leu Thr Asp         #       125                                                                   - Ala Asp Met Val Met Ser Phe Val Asn Leu Va - #l Glu His Asp Lys Glu         #   140                                                                       - Phe Phe His Pro Arg Tyr His His Arg Glu Ph - #e Arg Phe Asp Leu Ser         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Lys Ile Pro Glu Gly Glu Arg Val Thr Ala Al - #a Glu Phe Arg Ile Tyr         #               175                                                           - Lys Asp Tyr Ile Arg Glu Arg Phe Asp Asn Gl - #u Thr Phe Gln Ile Thr         #           190                                                               - Val Tyr Gln Val Leu Gln Glu His Ser Gly Ar - #g Glu Ser Asp Leu Phe         #       205                                                                   - Leu Leu Asp Ser Arg Thr Ile Trp Ala Ser Gl - #u Glu Gly Trp Leu Val         #   220                                                                       - Phe Asp Ile Thr Ala Thr Ser Asn His Trp Va - #l Val Asn Pro Arg His         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Asn Leu Gly Leu Gln Leu Ser Val Glu Thr Le - #u Asp Gly Gln Ser Ile         #               255                                                           - Asn Pro Lys Leu Ala Gly Leu Ile Gly Arg Hi - #s Gly Pro Gln Asn Lys         #           270                                                               - Gln Pro Phe Met Val Ala Phe Phe Lys Ala Th - #r Glu Val His Leu Arg         #       285                                                                   - Ser Ile Arg Ser Thr Gly Gly Lys Gln Arg Se - #r Gln Asn Arg Ser Lys         #   300                                                                       - Thr Pro Lys Asn Gln Glu Ala Leu Arg Met Al - #a Ser Val Ala Glu Asn         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Ser Ser Ser Asp Gln Arg Gln Ala Cys Lys Ly - #s His Glu Leu Tyr Val         #               335                                                           - Ser Phe Arg Asp Leu Gly Trp Gln Asp Trp Il - #e Ile Ala Pro Glu Gly         #           350                                                               - Tyr Ala Ala Tyr Tyr Cys Glu Gly Glu Cys Al - #a Phe Pro Leu Asn Ser         #       365                                                                   - Tyr Met Asn Ala Thr Asn His Ala Ile Val Gl - #n Thr Leu Val His Phe         #   380                                                                       - Ile Asn Pro Asp Thr Val Pro Lys Pro Cys Cy - #s Ala Pro Thr Gln Leu         385                 3 - #90                 3 - #95                 4 -       #00                                                                           - Asn Ala Ile Ser Val Leu Tyr Phe Asp Asp Se - #r Ser Asn Val Ile Leu         #               415                                                           - Lys Lys Tyr Arg Asn Met Val Val Arg Ala Cy - #s Gly Cys His                 #           430                                                               - (2) INFORMATION FOR SEQ ID NO:5:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 1723 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Homo sapi - #ens                                                (F) TISSUE TYPE: HIPPOC - #AMPUS                                    -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 490..1696                                             #/function= "OSTEOGENIC PROTEIN"                                                             /product=- # "hOP2-PP"                                         #"hOP2 (cDNA)" /note=                                                         -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                 - GGCGCCGGCA GAGCAGGAGT GGCTGGAGGA GCTGTGGTTG GAGCAGGAGG TG - #GCACGGCA         60                                                                          - GGGCTGGAGG GCTCCCTATG AGTGGCGGAG ACGGCCCAGG AGGCGCTGGA GC - #AACAGCTC        120                                                                          - CCACACCGCA CCAAGCGGTG GCTGCAGGAG CTCGCCCATC GCCCCTGCGC TG - #CTCGGACC        180                                                                          - GCGGCCACAG CCGGACTGGC GGGTACGGCG GCGACAGAGG CATTGGCCGA GA - #GTCCCAGT        240                                                                          - CCGCAGAGTA GCCCCGGCCT CGAGGCGGTG GCGTCCCGGT CCTCTCCGTC CA - #GGAGCCAG        300                                                                          - GACAGGTGTC GCGCGGCGGG GCTCCAGGGA CCGCGCCTGA GGCCGGCTGC CC - #GCCCGTCC        360                                                                          - CGCCCCGCCC CGCCGCCCGC CGCCCGCCGA GCCCAGCCTC CTTGCCGTCG GG - #GCGTCCCC        420                                                                          - AGGCCCTGGG TCGGCCGCGG AGCCGATGCG CGCCCGCTGA GCGCCCCAGC TG - #AGCGCCCC        480                                                                          - CGGCCTGCC ATG ACC GCG CTC CCC GGC CCG CTC TGG - # CTC CTG GGC CTG            528                                                                          #Gly Pro Leu Trp Leu Leu Gly Leu                                              #         10                                                                  - GCG CTA TGC GCG CTG GGC GGG GGC GGC CCC GG - #C CTG CGA CCC CCG CCC          576                                                                          Ala Leu Cys Ala Leu Gly Gly Gly Gly Pro Gl - #y Leu Arg Pro Pro Pro           #     25                                                                      - GGC TGT CCC CAG CGA CGT CTG GGC GCG CGC GA - #G CGC CGG GAC GTG CAG          624                                                                          Gly Cys Pro Gln Arg Arg Leu Gly Ala Arg Gl - #u Arg Arg Asp Val Gln           # 45                                                                          - CGC GAG ATC CTG GCG GTG CTC GGG CTG CCT GG - #G CGG CCC CGG CCC CGC          672                                                                          Arg Glu Ile Leu Ala Val Leu Gly Leu Pro Gl - #y Arg Pro Arg Pro Arg           #                 60                                                          - GCG CCA CCC GCC GCC TCC CGG CTG CCC GCG TC - #C GCG CCG CTC TTC ATG          720                                                                          Ala Pro Pro Ala Ala Ser Arg Leu Pro Ala Se - #r Ala Pro Leu Phe Met           #             75                                                              - CTG GAC CTG TAC CAC GCC ATG GCC GGC GAC GA - #C GAC GAG GAC GGC GCG          768                                                                          Leu Asp Leu Tyr His Ala Met Ala Gly Asp As - #p Asp Glu Asp Gly Ala           #         90                                                                  - CCC GCG GAG CGG CGC CTG GGC CGC GCC GAC CT - #G GTC ATG AGC TTC GTT          816                                                                          Pro Ala Glu Arg Arg Leu Gly Arg Ala Asp Le - #u Val Met Ser Phe Val           #    105                                                                      - AAC ATG GTG GAG CGA GAC CGT GCC CTG GGC CA - #C CAG GAG CCC CAT TGG          864                                                                          Asn Met Val Glu Arg Asp Arg Ala Leu Gly Hi - #s Gln Glu Pro His Trp           110                 1 - #15                 1 - #20                 1 -       #25                                                                           - AAG GAG TTC CGC TTT GAC CTG ACC CAG ATC CC - #G GCT GGG GAG GCG GTC          912                                                                          Lys Glu Phe Arg Phe Asp Leu Thr Gln Ile Pr - #o Ala Gly Glu Ala Val           #               140                                                           - ACA GCT GCG GAG TTC CGG ATT TAC AAG GTG CC - #C AGC ATC CAC CTG CTC          960                                                                          Thr Ala Ala Glu Phe Arg Ile Tyr Lys Val Pr - #o Ser Ile His Leu Leu           #           155                                                               - AAC AGG ACC CTC CAC GTC AGC ATG TTC CAG GT - #G GTC CAG GAG CAG TCC         1008                                                                          Asn Arg Thr Leu His Val Ser Met Phe Gln Va - #l Val Gln Glu Gln Ser           #       170                                                                   - AAC AGG GAG TCT GAC TTG TTC TTT TTG GAT CT - #T CAG ACG CTC CGA GCT         1056                                                                          Asn Arg Glu Ser Asp Leu Phe Phe Leu Asp Le - #u Gln Thr Leu Arg Ala           #   185                                                                       - GGA GAC GAG GGC TGG CTG GTG CTG GAT GTC AC - #A GCA GCC AGT GAC TGC         1104                                                                          Gly Asp Glu Gly Trp Leu Val Leu Asp Val Th - #r Ala Ala Ser Asp Cys           190                 1 - #95                 2 - #00                 2 -       #05                                                                           - TGG TTG CTG AAG CGT CAC AAG GAC CTG GGA CT - #C CGC CTC TAT GTG GAG         1152                                                                          Trp Leu Leu Lys Arg His Lys Asp Leu Gly Le - #u Arg Leu Tyr Val Glu           #               220                                                           - ACT GAG GAC GGG CAC AGC GTG GAT CCT GGC CT - #G GCC GGC CTG CTG GGT         1200                                                                          Thr Glu Asp Gly His Ser Val Asp Pro Gly Le - #u Ala Gly Leu Leu Gly           #           235                                                               - CAA CGG GCC CCA CGC TCC CAA CAG CCT TTC GT - #G GTC ACT TTC TTC AGG         1248                                                                          Gln Arg Ala Pro Arg Ser Gln Gln Pro Phe Va - #l Val Thr Phe Phe Arg           #       250                                                                   - GCC AGT CCG AGT CCC ATC CGC ACC CCT CGG GC - #A GTG AGG CCA CTG AGG         1296                                                                          Ala Ser Pro Ser Pro Ile Arg Thr Pro Arg Al - #a Val Arg Pro Leu Arg           #   265                                                                       - AGG AGG CAG CCG AAG AAA AGC AAC GAG CTG CC - #G CAG GCC AAC CGA CTC         1344                                                                          Arg Arg Gln Pro Lys Lys Ser Asn Glu Leu Pr - #o Gln Ala Asn Arg Leu           270                 2 - #75                 2 - #80                 2 -       #85                                                                           - CCA GGG ATC TTT GAT GAC GTC CAC GGC TCC CA - #C GGC CGG CAG GTC TGC         1392                                                                          Pro Gly Ile Phe Asp Asp Val His Gly Ser Hi - #s Gly Arg Gln Val Cys           #               300                                                           - CGT CGG CAC GAG CTC TAC GTC AGC TTC CAG GA - #C CTC GGC TGG CTG GAC         1440                                                                          Arg Arg His Glu Leu Tyr Val Ser Phe Gln As - #p Leu Gly Trp Leu Asp           #           315                                                               - TGG GTC ATC GCT CCC CAA GGC TAC TCG GCC TA - #T TAC TGT GAG GGG GAG         1488                                                                          Trp Val Ile Ala Pro Gln Gly Tyr Ser Ala Ty - #r Tyr Cys Glu Gly Glu           #       330                                                                   - TGC TCC TTC CCA CTG GAC TCC TGC ATG AAT GC - #C ACC AAC CAC GCC ATC         1536                                                                          Cys Ser Phe Pro Leu Asp Ser Cys Met Asn Al - #a Thr Asn His Ala Ile           #   345                                                                       - CTG CAG TCC CTG GTG CAC CTG ATG AAG CCA AA - #C GCA GTC CCC AAG GCG         1584                                                                          Leu Gln Ser Leu Val His Leu Met Lys Pro As - #n Ala Val Pro Lys Ala           350                 3 - #55                 3 - #60                 3 -       #65                                                                           - TGC TGT GCA CCC ACC AAG CTG AGC GCC ACC TC - #T GTG CTC TAC TAT GAC         1632                                                                          Cys Cys Ala Pro Thr Lys Leu Ser Ala Thr Se - #r Val Leu Tyr Tyr Asp           #               380                                                           - AGC AGC AAC AAC GTC ATC CTG CGC AAA GCC CG - #C AAC ATG GTG GTC AAG         1680                                                                          Ser Ser Asn Asn Val Ile Leu Arg Lys Ala Ar - #g Asn Met Val Val Lys           #           395                                                               - GCC TGC GGC TGC CAC T GAGTCAGCCC GCCCAGCCCT ACTG - #CAG                     #1723                                                                         Ala Cys Gly Cys His                                                                   400                                                                   - (2) INFORMATION FOR SEQ ID NO:6:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 402 amino                                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                 - Met Thr Ala Leu Pro Gly Pro Leu Trp Leu Le - #u Gly Leu Ala Leu Cys         #                 15                                                          - Ala Leu Gly Gly Gly Gly Pro Gly Leu Arg Pr - #o Pro Pro Gly Cys Pro         #             30                                                              - Gln Arg Arg Leu Gly Ala Arg Glu Arg Arg As - #p Val Gln Arg Glu Ile         #         45                                                                  - Leu Ala Val Leu Gly Leu Pro Gly Arg Pro Ar - #g Pro Arg Ala Pro Pro         #     60                                                                      - Ala Ala Ser Arg Leu Pro Ala Ser Ala Pro Le - #u Phe Met Leu Asp Leu         # 80                                                                          - Tyr His Ala Met Ala Gly Asp Asp Asp Glu As - #p Gly Ala Pro Ala Glu         #                 95                                                          - Arg Arg Leu Gly Arg Ala Asp Leu Val Met Se - #r Phe Val Asn Met Val         #           110                                                               - Glu Arg Asp Arg Ala Leu Gly His Gln Glu Pr - #o His Trp Lys Glu Phe         #       125                                                                   - Arg Phe Asp Leu Thr Gln Ile Pro Ala Gly Gl - #u Ala Val Thr Ala Ala         #   140                                                                       - Glu Phe Arg Ile Tyr Lys Val Pro Ser Ile Hi - #s Leu Leu Asn Arg Thr         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Leu His Val Ser Met Phe Gln Val Val Gln Gl - #u Gln Ser Asn Arg Glu         #               175                                                           - Ser Asp Leu Phe Phe Leu Asp Leu Gln Thr Le - #u Arg Ala Gly Asp Glu         #           190                                                               - Gly Trp Leu Val Leu Asp Val Thr Ala Ala Se - #r Asp Cys Trp Leu Leu         #       205                                                                   - Lys Arg His Lys Asp Leu Gly Leu Arg Leu Ty - #r Val Glu Thr Glu Asp         #   220                                                                       - Gly His Ser Val Asp Pro Gly Leu Ala Gly Le - #u Leu Gly Gln Arg Ala         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Pro Arg Ser Gln Gln Pro Phe Val Val Thr Ph - #e Phe Arg Ala Ser Pro         #               255                                                           - Ser Pro Ile Arg Thr Pro Arg Ala Val Arg Pr - #o Leu Arg Arg Arg Gln         #           270                                                               - Pro Lys Lys Ser Asn Glu Leu Pro Gln Ala As - #n Arg Leu Pro Gly Ile         #       285                                                                   - Phe Asp Asp Val His Gly Ser His Gly Arg Gl - #n Val Cys Arg Arg His         #   300                                                                       - Glu Leu Tyr Val Ser Phe Gln Asp Leu Gly Tr - #p Leu Asp Trp Val Ile         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Ala Pro Gln Gly Tyr Ser Ala Tyr Tyr Cys Gl - #u Gly Glu Cys Ser Phe         #               335                                                           - Pro Leu Asp Ser Cys Met Asn Ala Thr Asn Hi - #s Ala Ile Leu Gln Ser         #           350                                                               - Leu Val His Leu Met Lys Pro Asn Ala Val Pr - #o Lys Ala Cys Cys Ala         #       365                                                                   - Pro Thr Lys Leu Ser Ala Thr Ser Val Leu Ty - #r Tyr Asp Ser Ser Asn         #   380                                                                       - Asn Val Ile Leu Arg Lys Ala Arg Asn Met Va - #l Val Lys Ala Cys Gly         385                 3 - #90                 3 - #95                 4 -       #00                                                                           - Cys His                                                                     - (2) INFORMATION FOR SEQ ID NO:7:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 1926 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: MURIDAE                                                         (F) TISSUE TYPE: EMBRYO                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 93..1289                                              #/function= "OSTEOGENIC PROTEIN"                                                             /product=- # "mOP2-PP"                                         #"mOP2 cDNA"   /note=                                                         -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                 - GCCAGGCACA GGTGCGCCGT CTGGTCCTCC CCGTCTGGCG TCAGCCGAGC CC - #GACCAGCT         60                                                                          - ACCAGTGGAT GCGCGCCGGC TGAAAGTCCG AG ATG GCT ATG CGT - # CCC GGG CCA          113                                                                          #Met Ala Met Arg Pro Gly Pro                                                  #  1               5                                                          - CTC TGG CTA TTG GGC CTT GCT CTG TGC GCG CT - #G GGA GGC GGC CAC GGT          161                                                                          Leu Trp Leu Leu Gly Leu Ala Leu Cys Ala Le - #u Gly Gly Gly His Gly           #         20                                                                  - CCG CGT CCC CCG CAC ACC TGT CCC CAG CGT CG - #C CTG GGA GCG CGC GAG          209                                                                          Pro Arg Pro Pro His Thr Cys Pro Gln Arg Ar - #g Leu Gly Ala Arg Glu           #     35                                                                      - CGC CGC GAC ATG CAG CGT GAA ATC CTG GCG GT - #G CTC GGG CTA CCG GGA          257                                                                          Arg Arg Asp Met Gln Arg Glu Ile Leu Ala Va - #l Leu Gly Leu Pro Gly           # 55                                                                          - CGG CCC CGA CCC CGT GCA CAA CCC GCC GCT GC - #C CGG CAG CCA GCG TCC          305                                                                          Arg Pro Arg Pro Arg Ala Gln Pro Ala Ala Al - #a Arg Gln Pro Ala Ser           #                 70                                                          - GCG CCC CTC TTC ATG TTG GAC CTA TAC CAC GC - #C ATG ACC GAT GAC GAC          353                                                                          Ala Pro Leu Phe Met Leu Asp Leu Tyr His Al - #a Met Thr Asp Asp Asp           #             85                                                              - GAC GGC GGG CCA CCA CAG GCT CAC TTA GGC CG - #T GCC GAC CTG GTC ATG          401                                                                          Asp Gly Gly Pro Pro Gln Ala His Leu Gly Ar - #g Ala Asp Leu Val Met           #        100                                                                  - AGC TTC GTC AAC ATG GTG GAA CGC GAC CGT AC - #C CTG GGC TAC CAG GAG          449                                                                          Ser Phe Val Asn Met Val Glu Arg Asp Arg Th - #r Leu Gly Tyr Gln Glu           #   115                                                                       - CCA CAC TGG AAG GAA TTC CAC TTT GAC CTA AC - #C CAG ATC CCT GCT GGG          497                                                                          Pro His Trp Lys Glu Phe His Phe Asp Leu Th - #r Gln Ile Pro Ala Gly           120                 1 - #25                 1 - #30                 1 -       #35                                                                           - GAG GCT GTC ACA GCT GCT GAG TTC CGG ATC TA - #C AAA GAA CCC AGC ACC          545                                                                          Glu Ala Val Thr Ala Ala Glu Phe Arg Ile Ty - #r Lys Glu Pro Ser Thr           #               150                                                           - CAC CCG CTC AAC ACA ACC CTC CAC ATC AGC AT - #G TTC GAA GTG GTC CAA          593                                                                          His Pro Leu Asn Thr Thr Leu His Ile Ser Me - #t Phe Glu Val Val Gln           #           165                                                               - GAG CAC TCC AAC AGG GAG TCT GAC TTG TTC TT - #T TTG GAT CTT CAG ACG          641                                                                          Glu His Ser Asn Arg Glu Ser Asp Leu Phe Ph - #e Leu Asp Leu Gln Thr           #       180                                                                   - CTC CGA TCT GGG GAC GAG GGC TGG CTG GTG CT - #G GAC ATC ACA GCA GCC          689                                                                          Leu Arg Ser Gly Asp Glu Gly Trp Leu Val Le - #u Asp Ile Thr Ala Ala           #   195                                                                       - AGT GAC CGA TGG CTG CTG AAC CAT CAC AAG GA - #C CTG GGA CTC CGC CTC          737                                                                          Ser Asp Arg Trp Leu Leu Asn His His Lys As - #p Leu Gly Leu Arg Leu           200                 2 - #05                 2 - #10                 2 -       #15                                                                           - TAT GTG GAA ACC GCG GAT GGG CAC AGC ATG GA - #T CCT GGC CTG GCT GGT          785                                                                          Tyr Val Glu Thr Ala Asp Gly His Ser Met As - #p Pro Gly Leu Ala Gly           #               230                                                           - CTG CTT GGA CGA CAA GCA CCA CGC TCC AGA CA - #G CCT TTC ATG GTA ACC          833                                                                          Leu Leu Gly Arg Gln Ala Pro Arg Ser Arg Gl - #n Pro Phe Met Val Thr           #           245                                                               - TTC TTC AGG GCC AGC CAG AGT CCT GTG CGG GC - #C CCT CGG GCA GCG AGA          881                                                                          Phe Phe Arg Ala Ser Gln Ser Pro Val Arg Al - #a Pro Arg Ala Ala Arg           #       260                                                                   - CCA CTG AAG AGG AGG CAG CCA AAG AAA ACG AA - #C GAG CTT CCG CAC CCC          929                                                                          Pro Leu Lys Arg Arg Gln Pro Lys Lys Thr As - #n Glu Leu Pro His Pro           #   275                                                                       - AAC AAA CTC CCA GGG ATC TTT GAT GAT GGC CA - #C GGT TCC CGC GGC AGA          977                                                                          Asn Lys Leu Pro Gly Ile Phe Asp Asp Gly Hi - #s Gly Ser Arg Gly Arg           280                 2 - #85                 2 - #90                 2 -       #95                                                                           - GAG GTT TGC CGC AGG CAT GAG CTC TAC GTC AG - #C TTC CGT GAC CTT GGC         1025                                                                          Glu Val Cys Arg Arg His Glu Leu Tyr Val Se - #r Phe Arg Asp Leu Gly           #               310                                                           - TGG CTG GAC TGG GTC ATC GCC CCC CAG GGC TA - #C TCT GCC TAT TAC TGT         1073                                                                          Trp Leu Asp Trp Val Ile Ala Pro Gln Gly Ty - #r Ser Ala Tyr Tyr Cys           #           325                                                               - GAG GGG GAG TGT GCT TTC CCA CTG GAC TCC TG - #T ATG AAC GCC ACC AAC         1121                                                                          Glu Gly Glu Cys Ala Phe Pro Leu Asp Ser Cy - #s Met Asn Ala Thr Asn           #       340                                                                   - CAT GCC ATC TTG CAG TCT CTG GTG CAC CTG AT - #G AAG CCA GAT GTT GTC         1169                                                                          His Ala Ile Leu Gln Ser Leu Val His Leu Me - #t Lys Pro Asp Val Val           #   355                                                                       - CCC AAG GCA TGC TGT GCA CCC ACC AAA CTG AG - #T GCC ACC TCT GTG CTG         1217                                                                          Pro Lys Ala Cys Cys Ala Pro Thr Lys Leu Se - #r Ala Thr Ser Val Leu           360                 3 - #65                 3 - #70                 3 -       #75                                                                           - TAC TAT GAC AGC AGC AAC AAT GTC ATC CTG CG - #T AAA CAC CGT AAC ATG         1265                                                                          Tyr Tyr Asp Ser Ser Asn Asn Val Ile Leu Ar - #g Lys His Arg Asn Met           #               390                                                           - GTG GTC AAG GCC TGT GGC TGC CAC TGAGGCCCCG CC - #CAGCATCC TGCTTCTACT        1319                                                                          Val Val Lys Ala Cys Gly Cys His                                                           395                                                               - ACCTTACCAT CTGGCCGGGC CCCTCTCCAG AGGCAGAAAC CCTTCTATGT TA - #TCATAGCT       1379                                                                          - CAGACAGGGG CAATGGGAGG CCCTTCACTT CCCCTGGCCA CTTCCTGCTA AA - #ATTCTGGT       1439                                                                          - CTTTCCCAGT TCCTCTGTCC TTCATGGGGT TTCGGGGCTA TCACCCCGCC CT - #CTCCATCC       1499                                                                          - TCCTACCCCA AGCATAGACT GAATGCACAC AGCATCCCAG AGCTATGCTA AC - #TGAGAGGT       1559                                                                          - CTGGGGTCAG CACTGAAGGC CCACATGAGG AAGACTGATC CTTGGCCATC CT - #CAGCCCAC       1619                                                                          - AATGGCAAAT TCTGGATGGT CTAAGAAGGC CCTGGAATTC TAAACTAGAT GA - #TCTGGGCT       1679                                                                          - CTCTGCACCA TTCATTGTGG CAGTTGGGAC ATTTTTAGGT ATAACAGACA CA - #TACACTTA       1739                                                                          - GATCAATGCA TCGCTGTACT CCTTGAAATC AGAGCTAGCT TGTTAGAAAA AG - #AATCAGAG       1799                                                                          - CCAGGTATAG CGGTGCATGT CATTAATCCC AGCGCTAAAG AGACAGAGAC AG - #GAGAATCT       1859                                                                          - CTGTGAGTTC AAGGCCACAT AGAAAGAGCC TGTCTCGGGA GCAGGAAAAA AA - #AAAAAAAC       1919                                                                          #        1926                                                                 - (2) INFORMATION FOR SEQ ID NO:8:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 399 amino                                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                 - Met Ala Met Arg Pro Gly Pro Leu Trp Leu Le - #u Gly Leu Ala Leu Cys         #                 15                                                          - Ala Leu Gly Gly Gly His Gly Pro Arg Pro Pr - #o His Thr Cys Pro Gln         #             30                                                              - Arg Arg Leu Gly Ala Arg Glu Arg Arg Asp Me - #t Gln Arg Glu Ile Leu         #         45                                                                  - Ala Val Leu Gly Leu Pro Gly Arg Pro Arg Pr - #o Arg Ala Gln Pro Ala         #     60                                                                      - Ala Ala Arg Gln Pro Ala Ser Ala Pro Leu Ph - #e Met Leu Asp Leu Tyr         # 80                                                                          - His Ala Met Thr Asp Asp Asp Asp Gly Gly Pr - #o Pro Gln Ala His Leu         #                 95                                                          - Gly Arg Ala Asp Leu Val Met Ser Phe Val As - #n Met Val Glu Arg Asp         #           110                                                               - Arg Thr Leu Gly Tyr Gln Glu Pro His Trp Ly - #s Glu Phe His Phe Asp         #       125                                                                   - Leu Thr Gln Ile Pro Ala Gly Glu Ala Val Th - #r Ala Ala Glu Phe Arg         #   140                                                                       - Ile Tyr Lys Glu Pro Ser Thr His Pro Leu As - #n Thr Thr Leu His Ile         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Ser Met Phe Glu Val Val Gln Glu His Ser As - #n Arg Glu Ser Asp Leu         #               175                                                           - Phe Phe Leu Asp Leu Gln Thr Leu Arg Ser Gl - #y Asp Glu Gly Trp Leu         #           190                                                               - Val Leu Asp Ile Thr Ala Ala Ser Asp Arg Tr - #p Leu Leu Asn His His         #       205                                                                   - Lys Asp Leu Gly Leu Arg Leu Tyr Val Glu Th - #r Ala Asp Gly His Ser         #   220                                                                       - Met Asp Pro Gly Leu Ala Gly Leu Leu Gly Ar - #g Gln Ala Pro Arg Ser         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Arg Gln Pro Phe Met Val Thr Phe Phe Arg Al - #a Ser Gln Ser Pro Val         #               255                                                           - Arg Ala Pro Arg Ala Ala Arg Pro Leu Lys Ar - #g Arg Gln Pro Lys Lys         #           270                                                               - Thr Asn Glu Leu Pro His Pro Asn Lys Leu Pr - #o Gly Ile Phe Asp Asp         #       285                                                                   - Gly His Gly Ser Arg Gly Arg Glu Val Cys Ar - #g Arg His Glu Leu Tyr         #   300                                                                       - Val Ser Phe Arg Asp Leu Gly Trp Leu Asp Tr - #p Val Ile Ala Pro Gln         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Gly Tyr Ser Ala Tyr Tyr Cys Glu Gly Glu Cy - #s Ala Phe Pro Leu Asp         #               335                                                           - Ser Cys Met Asn Ala Thr Asn His Ala Ile Le - #u Gln Ser Leu Val His         #           350                                                               - Leu Met Lys Pro Asp Val Val Pro Lys Ala Cy - #s Cys Ala Pro Thr Lys         #       365                                                                   - Leu Ser Ala Thr Ser Val Leu Tyr Tyr Asp Se - #r Ser Asn Asn Val Ile         #   380                                                                       - Leu Arg Lys His Arg Asn Met Val Val Lys Al - #a Cys Gly Cys His             385                 3 - #90                 3 - #95                           - (2) INFORMATION FOR SEQ ID NO:9:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 399 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Protein                                                         (B) LOCATION: 1..399                                                #/note= "PRE-PRO-OP3 (MOUSE)"ON:                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                 - Met Ala Ala Arg Pro Gly Leu Leu Trp Leu Le - #u Gly Leu Ala Leu Cys         #                15                                                           - Val Leu Gly Gly Gly His Leu Ser His Pro Pr - #o His Val Phe Pro Gln         #            30                                                               - Arg Arg Leu Gly Val Arg Glu Pro Arg Asp Me - #t Gln Arg Glu Ile Arg         #        45                                                                   - Glu Val Leu Gly Leu Ala Gly Arg Pro Arg Se - #r Arg Ala Pro Val Gly         #    60                                                                       - Ala Ala Gln Gln Pro Ala Ser Ala Pro Leu Ph - #e Met Leu Asp Leu Tyr         #80                                                                           - Arg Ala Met Thr Asp Asp Ser Gly Gly Gly Th - #r Pro Gln Pro His Leu         #                95                                                           - Asp Arg Ala Asp Leu Ile Met Ser Phe Val As - #n Ile Val Glu Arg Asp         #           110                                                               - Arg Thr Leu Gly Tyr Gln Glu Pro His Trp Ly - #s Glu Phe His Phe Asp         #       125                                                                   - Leu Thr Gln Ile Pro Ala Gly Glu Ala Val Th - #r Ala Ala Glu Phe Arg         #   140                                                                       - Ile Tyr Lys Glu Pro Ser Thr His Pro Leu As - #n Thr Thr Leu His Ile         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Ser Met Phe Glu Val Val Gln Glu His Ser As - #n Arg Glu Ser Asp Leu         #               175                                                           - Phe Phe Leu Asp Leu Gln Thr Leu Arg Ser Gl - #y Asp Glu Gly Trp Leu         #           190                                                               - Val Leu Asp Ile Thr Ala Ala Ser Asp Arg Tr - #p Leu Leu Asn His His         #       205                                                                   - Lys Asp Leu Gly Leu Arg Leu Tyr Val Glu Th - #r Glu Asp Gly His Ser         #   220                                                                       - Ile Asp Pro Gly Leu Ala Gly Leu Leu Gly Ar - #g Gln Ala Pro Arg Ser         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Arg Gln Pro Phe Met Val Gly Phe Phe Arg Al - #a Asn Gln Ser Pro Val         #               255                                                           - Arg Ala Pro Arg Thr Ala Arg Pro Leu Lys Ly - #s Lys Gln Leu Asn Gln         #           270                                                               - Ile Asn Gln Leu Pro His Ser Asn Lys His Le - #u Gly Ile Leu Asp Asp         #       285                                                                   - Gly His Gly Ser His Gly Arg Glu Val Cys Ar - #g Arg His Glu Leu Tyr         #   300                                                                       - Val Ser Phe Arg Asp Leu Gly Trp Leu Asp Se - #r Val Ile Ala Pro Gln         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Gly Tyr Ser Ala Tyr Tyr Cys Ala Gly Glu Cy - #s Ile Tyr Pro Leu Asn         #               335                                                           - Ser Cys Met Asn Ser Thr Asn His Ala Thr Me - #t Gln Ala Leu Val His         #           350                                                               - Leu Met Lys Pro Asp Ile Ile Pro Lys Val Cy - #s Cys Val Pro Thr Glu         #       365                                                                   - Leu Ser Ala Ile Ser Leu Leu Tyr Tyr Asp Ar - #g Asn Asn Asn Val Ile         #   380                                                                       - Leu Arg Arg Glu Arg Asn Met Val Val Gln Al - #a Cys Gly Cys His             385                 3 - #90                 3 - #95                           - (2) INFORMATION FOR SEQ ID NO:10:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 396 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Protein                                                         (B) LOCATION: 1..396                                                #/note= "PRE-PRO-BMP2 (HUMAN)"N:                                              -      (x) PUBLICATION INFORMATION:                                                     (A) AUTHORS: WOZNEY,                                                          (C) JOURNAL: SCIENCE                                                          (D) VOLUME: 242                                                               (F) PAGES: 1528-1534                                                          (G) DATE: 1988                                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                - Met Val Ala Gly Thr Arg Cys Leu Leu Ala Le - #u Leu Leu Pro Gln Val         #                15                                                           - Leu Leu Gly Gly Ala Ala Gly Leu Val Pro Gl - #u Leu Gly Arg Arg Lys         #            30                                                               - Phe Ala Ala Ala Ser Ser Gly Arg Pro Ser Se - #r Gln Pro Ser Asp Glu         #        45                                                                   - Val Leu Ser Glu Phe Glu Leu Arg Leu Leu Se - #r Met Phe Gly Leu Lys         #    60                                                                       - Gln Arg Pro Thr Pro Ser Arg Asp Ala Val Va - #l Pro Pro Tyr Met Leu         #80                                                                           - Asp Leu Tyr Arg Arg His Ser Gly Gln Pro Gl - #y Ser Pro Ala Pro Asp         #                95                                                           - His Arg Leu Glu Arg Ala Ala Ser Arg Ala As - #n Thr Val Arg Ser Phe         #           110                                                               - His His Glu Glu Ser Leu Glu Glu Leu Pro Gl - #u Thr Ser Gly Lys Thr         #       125                                                                   - Thr Arg Arg Phe Phe Phe Asn Leu Ser Ser Il - #e Pro Thr Glu Glu Phe         #   140                                                                       - Ile Thr Ser Ala Glu Leu Gln Val Phe Arg Gl - #u Gln Met Gln Asp Ala         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Leu Gly Asn Asn Ser Ser Phe His His Arg Il - #e Asn Ile Tyr Glu Ile         #               175                                                           - Ile Lys Pro Ala Thr Ala Asn Ser Lys Phe Pr - #o Val Thr Arg Leu Leu         #           190                                                               - Asp Thr Arg Leu Val Asn Gln Asn Ala Ser Ar - #g Trp Glu Ser Phe Asp         #       205                                                                   - Val Thr Pro Ala Val Met Arg Trp Thr Ala Gl - #n Gly His Ala Asn His         #   220                                                                       - Gly Phe Val Val Glu Val Ala His Leu Glu Gl - #u Lys Gln Gly Val Ser         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Lys Arg His Val Arg Ile Ser Arg Ser Leu Hi - #s Gln Asp Glu His Ser         #               255                                                           - Trp Ser Gln Ile Arg Pro Leu Leu Val Thr Ph - #e Gly His Asp Gly Lys         #           270                                                               - Gly His Pro Leu His Lys Arg Glu Lys Arg Gl - #n Ala Lys His Lys Gln         #       285                                                                   - Arg Lys Arg Leu Lys Ser Ser Cys Lys Arg Hi - #s Pro Leu Tyr Val Asp         #   300                                                                       - Phe Ser Asp Val Gly Trp Asn Asp Trp Ile Va - #l Ala Pro Pro Gly Tyr         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - His Ala Phe Tyr Cys His Gly Glu Cys Pro Ph - #e Pro Leu Ala Asp His         #               335                                                           - Leu Asn Ser Thr Asn His Ala Ile Val Gln Th - #r Leu Val Asn Ser Val         #           350                                                               - Asn Ser Lys Ile Pro Lys Ala Cys Cys Val Pr - #o Thr Glu Leu Ser Ala         #       365                                                                   - Ile Ser Met Leu Tyr Leu Asp Glu Asn Glu Ly - #s Val Val Leu Lys Asn         #   380                                                                       - Tyr Gln Asp Met Val Val Glu Gly Cys Gly Cy - #s Arg                         385                 3 - #90                 3 - #95                           - (2) INFORMATION FOR SEQ ID NO:11:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 408 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Protein                                                         (B) LOCATION: 1..408                                                #/note= "PRE-PRO-BMP4 (HUMAN)"N:                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                - Met Ile Pro Gly Asn Arg Met Leu Met Val Va - #l Leu Leu Cys Gln Val         #                15                                                           - Leu Leu Gly Gly Ala Ser His Ala Ser Leu Il - #e Pro Glu Thr Gly Lys         #            30                                                               - Lys Lys Val Ala Glu Ile Gln Gly His Ala Gl - #y Gly Arg Arg Ser Gly         #        45                                                                   - Gln Ser His Glu Leu Leu Arg Asp Phe Glu Al - #a Thr Leu Leu Gln Met         #    60                                                                       - Phe Gly Leu Arg Arg Arg Pro Gln Pro Ser Ly - #s Ser Ala Val Ile Pro         #80                                                                           - Asp Tyr Met Arg Asp Leu Tyr Arg Leu Gln Se - #r Gly Glu Glu Glu Glu         #                95                                                           - Glu Gln Ile His Ser Thr Gly Leu Glu Tyr Pr - #o Glu Arg Pro Ala Ser         #           110                                                               - Arg Ala Asn Thr Val Arg Ser Phe His His Gl - #u Glu His Leu Glu Asn         #       125                                                                   - Ile Pro Gly Thr Ser Glu Asn Ser Ala Phe Ar - #g Phe Leu Phe Asn Leu         #   140                                                                       - Ser Ser Ile Pro Glu Asn Glu Val Ile Ser Se - #r Ala Glu Leu Arg Leu         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Phe Arg Glu Gln Val Asp Gln Gly Pro Asp Tr - #p Glu Arg Gly Phe His         #               175                                                           - Arg Ile Asn Ile Tyr Glu Val Met Lys Pro Pr - #o Ala Glu Val Val Pro         #           190                                                               - Gly His Leu Ile Thr Arg Leu Leu Asp Thr Ar - #g Leu Val His His Asn         #       205                                                                   - Val Thr Arg Trp Glu Thr Phe Asp Val Ser Pr - #o Ala Val Leu Arg Trp         #   220                                                                       - Thr Arg Glu Lys Gln Pro Asn Tyr Gly Leu Al - #a Ile Glu Val Thr His         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Leu His Gln Thr Arg Thr His Gln Gly Gln Hi - #s Val Arg Ile Ser Arg         #               255                                                           - Ser Leu Pro Gln Gly Ser Gly Asn Trp Ala Gl - #n Leu Arg Pro Leu Leu         #           270                                                               - Val Thr Phe Gly His Asp Gly Arg Gly His Al - #a Leu Thr Arg Arg Arg         #       285                                                                   - Arg Ala Lys Arg Ser Pro Lys His His Ser Gl - #n Arg Ala Arg Lys Lys         #   300                                                                       - Asn Lys Asn Cys Arg Arg His Ser Leu Tyr Va - #l Asp Phe Ser Asp Val         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Gly Trp Asn Asp Trp Ile Val Ala Pro Pro Gl - #y Tyr Gln Ala Phe Tyr         #               335                                                           - Cys His Gly Asp Cys Pro Phe Pro Leu Ala As - #p His Leu Asn Ser Thr         #           350                                                               - Asn His Ala Ile Val Gln Thr Leu Val Asn Se - #r Val Asn Ser Ser Ile         #       365                                                                   - Pro Lys Ala Cys Cys Val Pro Thr Glu Leu Se - #r Ala Ile Ser Met Leu         #   380                                                                       - Tyr Leu Asp Glu Tyr Asp Lys Val Val Leu Ly - #s Asn Tyr Gln Glu Met         385                 3 - #90                 3 - #95                 4 -       #00                                                                           - Val Val Glu Gly Cys Gly Cys Arg                                                             405                                                           - (2) INFORMATION FOR SEQ ID NO:12:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 588 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Protein                                                         (B) LOCATION: 1..588                                                #/note= "PRE-PRO-DPP"NFORMATION:                                              -      (x) PUBLICATION INFORMATION:                                                     (A) AUTHORS: PADGETT,                                                         (C) JOURNAL: NATURE                                                           (D) VOLUME: 325                                                               (F) PAGES: 81-84                                                              (G) DATE: 1987                                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                - Met Arg Ala Trp Leu Leu Leu Leu Ala Val Le - #u Ala Thr Phe Gln Thr         #                15                                                           - Ile Val Arg Val Ala Ser Thr Glu Asp Ile Se - #r Gln Arg Phe Ile Ala         #            30                                                               - Ala Ile Ala Pro Val Ala Ala His Ile Pro Le - #u Ala Ser Ala Ser Gly         #        45                                                                   - Ser Gly Ser Gly Arg Ser Gly Ser Arg Ser Va - #l Gly Ala Ser Thr Ser         #    60                                                                       - Thr Ala Leu Ala Lys Ala Phe Asn Pro Phe Se - #r Glu Pro Ala Ser Phe         #80                                                                           - Ser Asp Ser Asp Lys Ser His Arg Ser Lys Th - #r Asn Lys Lys Pro Ser         #                95                                                           - Lys Ser Asp Ala Asn Arg Gln Phe Asn Glu Va - #l His Lys Pro Arg Thr         #           110                                                               - Asp Gln Leu Glu Asn Ser Lys Asn Lys Ser Ly - #s Gln Leu Val Asn Lys         #       125                                                                   - Pro Asn His Asn Lys Met Ala Val Lys Glu Gl - #n Arg Ser His His Lys         #   140                                                                       - Lys Ser His His His Arg Ser His Gln Pro Ly - #s Gln Ala Ser Ala Ser         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Thr Glu Ser His Gln Ser Ser Ser Ile Glu Se - #r Ile Phe Val Glu Glu         #               175                                                           - Pro Thr Leu Val Leu Asp Arg Glu Val Ala Se - #r Ile Asn Val Pro Ala         #           190                                                               - Asn Ala Lys Ala Ile Ile Ala Glu Gln Gly Pr - #o Ser Thr Tyr Ser Lys         #       205                                                                   - Glu Ala Leu Ile Lys Asp Lys Leu Lys Pro As - #p Pro Ser Thr Leu Val         #   220                                                                       - Glu Ile Glu Lys Ser Leu Leu Ser Leu Phe As - #n Met Lys Arg Pro Pro         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Lys Ile Asp Arg Ser Lys Ile Ile Ile Pro Gl - #u Pro Met Lys Lys Leu         #               255                                                           - Tyr Ala Glu Ile Asn Gly His Glu Leu Asp Se - #r Val Asn Ile Pro Lys         #           270                                                               - Pro Gly Leu Leu Thr Lys Ser Ala Asn Thr Va - #l Arg Ser Phe Thr His         #       285                                                                   - Lys Asp Ser Lys Ile Asp Asp Arg Phe Pro Hi - #s His His Arg Phe Arg         #   300                                                                       - Leu His Phe Asp Val Lys Ser Ile Pro Ala As - #p Glu Lys Leu Lys Ala         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Ala Glu Leu Gln Leu Thr Arg Asp Ala Leu Se - #r Gln Gln Val Val Ala         #               335                                                           - Ser Arg Ser Ser Ala Asn Arg Thr Arg Tyr Gl - #n Val Leu Val Tyr Asp         #           350                                                               - Ile Thr Arg Val Gly Val Arg Gly Gln Arg Gl - #u Pro Ser Tyr Leu Leu         #       365                                                                   - Leu Asp Thr Lys Thr Val Arg Leu Asn Ser Th - #r Asp Thr Val Ser Leu         #   380                                                                       - Asp Val Gln Pro Ala Val Asp Arg Trp Leu Al - #a Ser Pro Gln Arg Asn         385                 3 - #90                 3 - #95                 4 -       #00                                                                           - Tyr Gly Leu Leu Val Glu Val Arg Thr Val Ar - #g Ser Leu Lys Pro Ala         #               415                                                           - Pro His His His Val Arg Leu Arg Arg Ser Al - #a Asp Glu Ala His Glu         #           430                                                               - Arg Trp Gln His Lys Gln Pro Leu Leu Phe Th - #r Tyr Thr Asp Asp Gly         #       445                                                                   - Arg His Lys Ala Arg Ser Ile Arg Asp Val Se - #r Gly Gly Glu Gly Gly         #   460                                                                       - Gly Lys Gly Gly Arg Asn Lys Arg His Ala Ar - #g Arg Pro Thr Arg Arg         465                 4 - #70                 4 - #75                 4 -       #80                                                                           - Lys Asn His Asp Asp Thr Cys Arg Arg His Se - #r Leu Tyr Val Asp Phe         #               495                                                           - Ser Asp Val Gly Trp Asp Asp Trp Ile Val Al - #a Pro Leu Gly Tyr Asp         #           510                                                               - Ala Tyr Tyr Cys His Gly Lys Cys Pro Phe Pr - #o Leu Ala Asp His Phe         #       525                                                                   - Asn Ser Thr Asn His Ala Val Val Gln Thr Le - #u Val Asn Asn Met Asn         #   540                                                                       - Pro Gly Lys Val Pro Lys Ala Cys Cys Val Pr - #o Thr Gln Leu Asp Ser         545                 5 - #50                 5 - #55                 5 -       #60                                                                           - Val Ala Met Leu Tyr Leu Asn Asp Gln Ser Th - #r Val Val Leu Lys Asn         #               575                                                           - Tyr Gln Glu Met Thr Val Val Gly Cys Gly Cy - #s Arg                         #           585                                                               - (2) INFORMATION FOR SEQ ID NO:13:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 360 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Protein                                                         (B) LOCATION: 1..360                                                #/note= "PRE-PRO-VG1"NFORMATION:                                              -      (x) PUBLICATION INFORMATION:                                                     (A) AUTHORS: WEEKS,                                                           (C) JOURNAL: CELL                                                             (D) VOLUME: 51                                                                (F) PAGES: 861-867                                                            (G) DATE: 1987                                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                - Met Val Trp Leu Arg Leu Trp Ala Phe Leu Hi - #s Ile Leu Ala Ile Val         #                15                                                           - Thr Leu Asp Pro Glu Leu Lys Arg Arg Glu Gl - #u Leu Phe Leu Arg Ser         #            30                                                               - Leu Gly Phe Ser Ser Lys Pro Asn Pro Val Se - #r Pro Pro Pro Val Pro         #        45                                                                   - Ser Ile Leu Trp Arg Ile Phe Asn Gln Arg Me - #t Gly Ser Ser Ile Gln         #    60                                                                       - Lys Lys Lys Pro Asp Leu Cys Phe Val Glu Gl - #u Phe Asn Val Pro Gly         #80                                                                           - Ser Val Ile Arg Val Phe Pro Asp Gln Gly Ar - #g Phe Ile Ile Pro Tyr         #                95                                                           - Ser Asp Asp Ile His Pro Thr Gln Cys Leu Gl - #y Lys Arg Leu Phe Phe         #           110                                                               - Asn Ile Ser Ala Ile Glu Lys Glu Glu Arg Va - #l Thr Met Gly Ser Gly         #       125                                                                   - Ile Glu Val Gln Pro Glu His Leu Leu Arg Ly - #s Gly Ile Asp Leu Arg         #   140                                                                       - Leu Tyr Arg Thr Leu Gln Ile Thr Leu Lys Gl - #y Met Gly Arg Ser Lys         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Thr Ser Arg Lys Leu Leu Val Ala Gln Thr Ph - #e Arg Leu Leu His Lys         #               175                                                           - Ser Leu Phe Phe Asn Leu Thr Glu Ile Cys Gl - #n Ser Trp Gln Asp Pro         #           190                                                               - Leu Lys Asn Leu Gly Leu Val Leu Glu Ile Ph - #e Pro Lys Lys Glu Ser         #       205                                                                   - Ser Trp Met Ser Thr Ala Asn Asp Glu Cys Ly - #s Asp Ile Gln Thr Phe         #   220                                                                       - Leu Tyr Thr Ser Leu Leu Thr Val Thr Leu As - #n Pro Leu Arg Cys Lys         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Arg Pro Arg Arg Lys Arg Ser Tyr Ser Lys Le - #u Pro Phe Thr Ala Ser         #               255                                                           - Asn Ile Cys Lys Lys Arg His Leu Tyr Val Gl - #u Phe Lys Asp Val Gly         #           270                                                               - Trp Gln Asn Trp Val Ile Ala Pro Gln Gly Ty - #r Met Ala Asn Tyr Cys         #       285                                                                   - Tyr Gly Glu Cys Pro Tyr Pro Leu Thr Glu Il - #e Leu Asn Gly Ser Asn         #   300                                                                       - His Ala Ile Leu Gln Thr Leu Val His Ser Il - #e Glu Pro Glu Asp Ile         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Pro Leu Pro Cys Cys Val Pro Thr Lys Met Se - #r Pro Ile Ser Met Leu         #               335                                                           - Phe Tyr Asp Asn Asn Asp Asn Val Val Leu Ar - #g His Tyr Glu Asn Met         #           350                                                               - Ala Val Asp Glu Cys Gly Cys Arg                                             #       360                                                                   - (2) INFORMATION FOR SEQ ID NO:14:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 438 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Protein                                                         (B) LOCATION: 1..438                                                #/note= "PRE-PRO-VGR1"FORMATION:                                              -      (x) PUBLICATION INFORMATION:                                                     (A) AUTHORS: LYONS,                                                           (C) JOURNAL: Proc. Natl - #. Acad. Sci. U.S.A.                                (D) VOLUME: 86                                                                (F) PAGES: 4554-4558                                                          (G) DATE: 1989                                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                - Met Arg Lys Met Gln Lys Glu Ile Leu Ser Va - #l Leu Gly Pro Pro His         #                15                                                           - Arg Pro Arg Pro Leu His Gly Leu Gln Gln Pr - #o Gln Pro Pro Val Leu         #            30                                                               - Pro Pro Gln Gln Gln Gln Gln Gln Gln Gln Gl - #n Gln Thr Ala Asp Glu         #        45                                                                   - Glu Pro Pro Pro Gly Arg Leu Lys Ser Ala Pr - #o Leu Phe Met Leu Asp         #    60                                                                       - Leu Tyr Asn Ala Leu Ser Asn Asp Asp Glu Gl - #u Asp Gly Ala Ser Glu         #80                                                                           - Gly Val Gly Gln Glu Pro Gly Ser His Gly Gl - #y Ala Ser Ser Ser Gln         #                95                                                           - Leu Arg Gln Pro Ser Pro Gly Ala Ala His Se - #r Leu Asn Arg Lys Ser         #           110                                                               - Leu Leu Ala Pro Gly Pro Gly Gly Gly Ala Se - #r Pro Leu Thr Ser Ala         #       125                                                                   - Gln Asp Ser Ala Phe Leu Asn Asp Ala Asp Me - #t Val Met Ser Phe Val         #   140                                                                       - Asn Leu Val Gly Tyr Asp Lys Glu Phe Ser Pr - #o His Gln Arg His His         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Lys Glu Phe Lys Phe Asn Leu Ser Gln Ile Pr - #o Glu Gly Glu Ala Val         #               175                                                           - Thr Ala Ala Glu Phe Arg Val Tyr Lys Asp Cy - #s Val Val Gly Ser Phe         #           190                                                               - Lys Asn Gln Thr Phe Leu Ile Ser Ile Tyr Gl - #n Val Leu Gln Glu Ala         #       205                                                                   - Gln His Arg Asp Ser Asp Leu Phe Leu Leu As - #p Thr Arg Val Val Trp         #   220                                                                       - Ala Ser Glu Glu Gly Trp Leu Glu Phe Asp Il - #e Thr Ala Thr Ser Asn         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Leu Trp Val Val Ile Pro Gln His Asn Met Gl - #y Leu Gln Leu Ser Val         #               255                                                           - Val Thr Arg Asp Gly Leu His Val Asn Pro Ar - #g Ala Ala Gly Leu Val         #           270                                                               - Gly Arg Asp Gly Pro Tyr Asp Lys Gln Pro Ph - #e Met Val Ala Phe Phe         #       285                                                                   - Lys Val Ser Glu Val His Val Arg Thr Thr Ar - #g Ser Ala Ser Ser Arg         #   300                                                                       - Arg Arg Gln Gln Ser Arg Asn Arg Ser Thr Gl - #n Ser Gln Asp Val Ser         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Arg Gly Ser Gly Ser Ser Asp Tyr Asn Gly Se - #r Glu Leu Lys Thr Ala         #               335                                                           - Cys Lys Lys His Glu Leu Tyr Val Ser Phe Gl - #n Asp Leu Gly Trp Gln         #           350                                                               - Asp Trp Ile Ile Ala Pro Lys Gly Tyr Ala Al - #a Asn Tyr Cys Asp Gly         #       365                                                                   - Glu Cys Ser Phe Pro Leu Asn Ala His Met As - #n Ala Thr Asn His Ala         #   380                                                                       - Ile Val Gln Thr Leu Val His Leu Met Asn Pr - #o Glu Thr Val Pro Lys         385                 3 - #90                 3 - #95                 4 -       #00                                                                           - Pro Cys Cys Ala Pro Thr Lys Leu Asn Ala Il - #e Ser Val Leu Tyr Phe         #               415                                                           - Asp Asp Asn Ser Asn Val Ile Leu Lys Lys Ty - #r Arg Asn Met Val Val         #           430                                                               - Arg Ala Cys Gly Cys His                                                             435                                                                   - (2) INFORMATION FOR SEQ ID NO:15:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 372 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Protein                                                         (B) LOCATION: 1..372                                                #/note= "PRE-PRO-GDF-1"ORMATION:                                              -      (x) PUBLICATION INFORMATION:                                                     (A) AUTHORS: LEE,                                                             (B) TITLE: EXPRESSION O - #F GROWTH/DIFFERENTIATION FACTOR 1                  (C) JOURNAL: Proc. Natl - #. Acad. Sci. U.S.A.                                (D) VOLUME: 88                                                                (F) PAGES: 4250-4254                                                          (G) DATE: MAY-1991                                                  -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                - Met Pro Pro Pro Gln Gln Gly Pro Cys Gly Hi - #s His Leu Leu Leu Leu         #                15                                                           - Leu Ala Leu Leu Leu Pro Ser Leu Pro Leu Th - #r Arg Ala Pro Val Pro         #            30                                                               - Pro Gly Pro Ala Ala Ala Leu Leu Gln Ala Le - #u Gly Leu Arg Asp Glu         #        45                                                                   - Pro Gln Gly Ala Pro Arg Leu Arg Pro Val Pr - #o Pro Val Met Trp Arg         #    60                                                                       - Leu Phe Arg Arg Arg Asp Pro Gln Glu Thr Ar - #g Ser Gly Ser Arg Arg         #80                                                                           - Thr Ser Pro Gly Val Thr Leu Gln Pro Cys Hi - #s Val Glu Glu Leu Gly         #                95                                                           - Val Ala Gly Asn Ile Val Arg His Ile Pro As - #p Arg Gly Ala Pro Thr         #           110                                                               - Arg Ala Ser Glu Pro Val Ser Ala Ala Gly Hi - #s Cys Pro Glu Trp Thr         #       125                                                                   - Val Val Phe Asp Leu Ser Ala Val Glu Pro Al - #a Glu Arg Pro Ser Arg         #   140                                                                       - Ala Arg Leu Glu Leu Arg Phe Ala Ala Ala Al - #a Ala Ala Ala Pro Glu         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Gly Gly Trp Glu Leu Ser Val Ala Gln Ala Gl - #y Gln Gly Ala Gly Ala         #               175                                                           - Asp Pro Gly Pro Val Leu Leu Arg Gln Leu Va - #l Pro Ala Leu Gly Pro         #           190                                                               - Pro Val Arg Ala Glu Leu Leu Gly Ala Ala Tr - #p Ala Arg Asn Ala Ser         #       205                                                                   - Trp Pro Arg Ser Leu Arg Leu Ala Leu Ala Le - #u Arg Pro Arg Ala Pro         #   220                                                                       - Ala Ala Cys Ala Arg Leu Ala Glu Ala Ser Le - #u Leu Leu Val Thr Leu         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Asp Pro Arg Leu Cys His Pro Leu Ala Arg Pr - #o Arg Arg Asp Ala Glu         #               255                                                           - Pro Val Leu Gly Gly Gly Pro Gly Gly Ala Cy - #s Arg Ala Arg Arg Leu         #           270                                                               - Tyr Val Ser Phe Arg Glu Val Gly Trp His Ar - #g Trp Val Ile Ala Pro         #       285                                                                   - Arg Gly Phe Leu Ala Asn Tyr Cys Gln Gly Gl - #n Cys Ala Leu Pro Val         #   300                                                                       - Ala Leu Ser Gly Ser Gly Gly Pro Pro Ala Le - #u Asn His Ala Val Leu         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Arg Ala Leu Met His Ala Ala Ala Pro Gly Al - #a Ala Asp Leu Pro Cys         #               335                                                           - Cys Val Pro Ala Arg Leu Ser Pro Ile Ser Va - #l Leu Phe Phe Asp Asn         #           350                                                               - Ser Asp Asn Val Val Leu Arg Gln Tyr Glu As - #p Met Val Val Asp Glu         #       365                                                                   - Cys Gly Cys Arg                                                                 370                                                                       - (2) INFORMATION FOR SEQ ID NO:16:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 455 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Protein                                                         (B) LOCATION: 1..455                                                #/note= "PRE-PRO 60A"NFORMATION:                                              -      (x) PUBLICATION INFORMATION:                                                     (A) AUTHORS: WHARTON,                                                         (C) JOURNAL: Proc. Natl - #. Acad. Sci. U.S.A.                                (D) VOLUME: 88                                                                (F) PAGES: 9214-9218                                                          (G) DATE: 1991                                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                - Met Ser Gly Leu Arg Asn Thr Ser Glu Ala Va - #l Ala Val Leu Ala Ser         #                15                                                           - Leu Gly Leu Gly Met Val Leu Leu Met Phe Va - #l Ala Thr Thr Pro Pro         #            30                                                               - Ala Val Glu Ala Thr Gln Ser Gly Ile Tyr Il - #e Asp Asn Gly Lys Asp         #        45                                                                   - Gln Thr Ile Met His Arg Val Leu Ser Glu As - #p Asp Lys Leu Asp Val         #    60                                                                       - Ser Tyr Glu Ile Leu Glu Phe Leu Gly Ile Al - #a Glu Arg Pro Thr His         #80                                                                           - Leu Ser Ser His Gln Leu Ser Leu Arg Lys Se - #r Ala Pro Lys Phe Leu         #                95                                                           - Leu Asp Val Tyr His Arg Ile Thr Ala Glu Gl - #u Gly Leu Ser Asp Gln         #           110                                                               - Asp Glu Asp Asp Asp Tyr Glu Arg Gly His Ar - #g Ser Arg Arg Ser Ala         #       125                                                                   - Asp Leu Glu Glu Asp Glu Gly Glu Gln Gln Ly - #s Asn Phe Ile Thr Asp         #   140                                                                       - Leu Asp Lys Arg Ala Ile Asp Glu Ser Asp Il - #e Ile Met Thr Phe Leu         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Asn Lys Arg His His Asn Val Asp Glu Leu Ar - #g His Glu His Gly Arg         #               175                                                           - Arg Leu Trp Phe Asp Val Ser Asn Val Pro As - #n Asp Asn Tyr Leu Val         #           190                                                               - Met Ala Glu Leu Arg Ile Tyr Gln Asn Ala As - #n Glu Gly Lys Trp Leu         #       205                                                                   - Thr Ala Asn Arg Glu Phe Thr Ile Thr Val Ty - #r Ala Ile Gly Thr Gly         #   220                                                                       - Thr Leu Gly Gln His Thr Met Glu Pro Leu Se - #r Ser Val Asn Thr Thr         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Gly Asp Tyr Val Gly Trp Leu Glu Leu Asn Va - #l Thr Glu Gly Leu His         #               255                                                           - Glu Trp Leu Val Lys Ser Lys Asp Asn His Gl - #y Ile Tyr Ile Gly Ala         #           270                                                               - His Ala Val Asn Arg Pro Asp Arg Glu Val Ly - #s Leu Asp Asp Ile Gly         #       285                                                                   - Leu Ile His Arg Lys Val Asp Asp Glu Phe Gl - #n Pro Phe Met Ile Gly         #   300                                                                       - Phe Phe Arg Gly Pro Glu Leu Ile Lys Ala Th - #r Ala His Ser Ser His         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - His Arg Ser Lys Arg Ser Ala Ser His Pro Ar - #g Lys Arg Lys Lys Ser         #               335                                                           - Val Ser Pro Asn Asn Val Pro Leu Leu Glu Pr - #o Met Glu Ser Thr Arg         #           350                                                               - Ser Cys Gln Met Gln Thr Leu Tyr Ile Asp Ph - #e Lys Asp Leu Gly Trp         #       365                                                                   - His Asp Trp Ile Ile Ala Pro Glu Gly Tyr Gl - #y Ala Phe Tyr Cys Ser         #   380                                                                       - Gly Glu Cys Asn Phe Pro Leu Asn Ala His Me - #t Asn Ala Thr Asn His         385                 3 - #90                 3 - #95                 4 -       #00                                                                           - Ala Ile Val Gln Thr Leu Val His Leu Leu Gl - #u Pro Lys Lys Val Pro         #               415                                                           - Lys Pro Cys Cys Ala Pro Thr Arg Leu Gly Al - #a Leu Pro Val Leu Tyr         #           430                                                               - His Leu Asn Asp Glu Asn Val Asn Leu Lys Ly - #s Tyr Arg Asn Met Ile         #       445                                                                   - Val Lys Ser Cys Gly Cys His                                                 #   455                                                                       - (2) INFORMATION FOR SEQ ID NO:17:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 472 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Protein                                                         (B) LOCATION: 1..472                                                #/note= "PRE-PRO-BMP3"FORMATION:                                              -      (x) PUBLICATION INFORMATION:                                                     (A) AUTHORS: WOZNEY,                                                          (C) JOURNAL: SCIENCE                                                          (D) VOLUME: 242                                                               (F) PAGES: 1528-1534                                                          (G) DATE: 1988                                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                - Met Ala Gly Ala Ser Arg Leu Leu Phe Leu Tr - #p Leu Gly Cys Phe Cys         #                15                                                           - Val Ser Leu Ala Gln Gly Glu Arg Pro Lys Pr - #o Pro Phe Pro Glu Leu         #            30                                                               - Arg Lys Ala Val Pro Gly Asp Arg Thr Ala Gl - #y Gly Gly Pro Asp Ser         #        45                                                                   - Glu Leu Gln Pro Gln Asp Lys Val Ser Glu Hi - #s Met Leu Arg Leu Tyr         #    60                                                                       - Asp Arg Tyr Ser Thr Val Gln Ala Ala Arg Th - #r Pro Gly Ser Leu Glu         #80                                                                           - Gly Gly Ser Gln Pro Trp Arg Pro Arg Leu Le - #u Arg Glu Gly Asn Thr         #                95                                                           - Val Arg Ser Phe Arg Ala Ala Ala Ala Glu Th - #r Leu Glu Arg Lys Gly         #           110                                                               - Leu Tyr Ile Phe Asn Leu Thr Ser Leu Thr Ly - #s Ser Glu Asn Ile Leu         #       125                                                                   - Ser Ala Thr Leu Tyr Phe Cys Ile Gly Glu Le - #u Gly Asn Ile Ser Leu         #   140                                                                       - Ser Cys Pro Val Ser Gly Gly Cys Ser His Hi - #s Ala Gln Arg Lys His         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Ile Gln Ile Asp Leu Ser Ala Trp Thr Leu Ly - #s Phe Ser Arg Asn Gln         #               175                                                           - Ser Gln Leu Leu Gly His Leu Ser Val Asp Me - #t Ala Lys Ser His Arg         #           190                                                               - Asp Ile Met Ser Trp Leu Ser Lys Asp Ile Th - #r Gln Phe Leu Arg Lys         #       205                                                                   - Ala Lys Glu Asn Glu Glu Phe Leu Ile Gly Ph - #e Asn Ile Thr Ser Lys         #   220                                                                       - Gly Arg Gln Leu Pro Lys Arg Arg Leu Pro Ph - #e Pro Glu Pro Tyr Ile         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Leu Val Tyr Ala Asn Asp Ala Ala Ile Ser Gl - #u Pro Glu Ser Val Val         #               255                                                           - Ser Ser Leu Gln Gly His Arg Asn Phe Pro Th - #r Gly Thr Val Pro Lys         #           270                                                               - Trp Asp Ser His Ile Arg Ala Ala Leu Ser Il - #e Glu Arg Arg Lys Lys         #       285                                                                   - Arg Ser Thr Gly Val Leu Leu Pro Leu Gln As - #n Asn Glu Leu Pro Gly         #   300                                                                       - Ala Glu Tyr Gln Tyr Lys Lys Asp Glu Val Tr - #p Glu Glu Arg Lys Pro         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Tyr Lys Thr Leu Gln Ala Gln Ala Pro Glu Ly - #s Ser Lys Asn Lys Lys         #               335                                                           - Lys Gln Arg Lys Gly Pro His Arg Lys Ser Gl - #n Thr Leu Gln Phe Asp         #           350                                                               - Glu Gln Thr Leu Lys Lys Ala Arg Arg Lys Gl - #n Trp Ile Glu Pro Arg         #       365                                                                   - Asn Cys Ala Arg Arg Tyr Leu Lys Val Asp Ph - #e Ala Asp Ile Gly Trp         #   380                                                                       - Ser Glu Trp Ile Ile Ser Pro Lys Ser Phe As - #p Ala Tyr Tyr Cys Ser         385                 3 - #90                 3 - #95                 4 -       #00                                                                           - Gly Ala Cys Gln Phe Pro Met Pro Lys Ser Le - #u Lys Pro Ser Asn His         #               415                                                           - Ala Thr Ile Gln Ser Ile Val Arg Ala Val Gl - #y Val Val Pro Gly Ile         #           430                                                               - Pro Glu Pro Cys Cys Val Pro Glu Lys Met Se - #r Ser Leu Ser Ile Leu         #       445                                                                   - Phe Phe Asp Glu Asn Lys Asn Val Val Leu Ly - #s Val Tyr Pro Asn Met         #   460                                                                       - Thr Val Glu Ser Cys Ala Cys Arg                                             465                 4 - #70                                                   - (2) INFORMATION FOR SEQ ID NO:18:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 453 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Protein                                                         (B) LOCATION: 1..453                                                #/note= "PRE-PRO-BMP5 (HUMAN)"N:                                              -      (x) PUBLICATION INFORMATION:                                                     (A) AUTHORS: CELESTE,                                                         (C) JOURNAL: Proc. Natl - #. Acad. Sci. U.S.A.                                (D) VOLUME: 87                                                                (F) PAGES: 9843-9847                                                          (G) DATE: 1991                                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                - Met His Leu Thr Val Phe Leu Leu Lys Gly Il - #e Val Gly Phe Leu Trp         #                15                                                           - Ser Cys Trp Val Leu Val Gly Tyr Ala Lys Gl - #y Gly Leu Gly Asp Asn         #            30                                                               - His Val His Ser Ser Phe Ile Tyr Arg Arg Le - #u Arg Asn His Glu Arg         #        45                                                                   - Arg Glu Ile Gln Arg Glu Ile Leu Ser Ile Le - #u Gly Leu Pro His Arg         #    60                                                                       - Pro Arg Pro Phe Ser Pro Gly Lys Gln Ala Se - #r Ser Ala Pro Leu Phe         #80                                                                           - Met Leu Asp Leu Tyr Asn Ala Met Thr Asn Gl - #u Glu Asn Pro Glu Glu         #                95                                                           - Ser Glu Tyr Ser Val Arg Ala Ser Leu Ala Gl - #u Glu Thr Arg Gly Ala         #           110                                                               - Arg Lys Gly Tyr Pro Ala Ser Pro Asn Gly Ty - #r Pro Arg Arg Ile Gln         #       125                                                                   - Leu Ser Arg Thr Thr Pro Leu Thr Thr Gln Se - #r Pro Pro Leu Ala Ser         #   140                                                                       - Leu His Asp Thr Asn Phe Leu Asn Asp Ala As - #p Met Val Met Ser Phe         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Val Asn Leu Val Glu Arg Asp Lys Asp Phe Se - #r His Gln Arg Arg His         #               175                                                           - Tyr Lys Glu Arg Phe Asp Leu Thr Gln Ile Pr - #o His Gly Glu Ala Val         #           190                                                               - Thr Ala Ala Glu Phe Arg Ile Tyr Lys Asp Ar - #g Ser Asn Asn Arg Phe         #       205                                                                   - Glu Asn Glu Thr Ile Lys Ile Ser Ile Tyr Gl - #n Ile Ile Lys Glu Tyr         #   220                                                                       - Thr Asn Arg Asp Ala Asp Leu Phe Leu Leu As - #p Thr Arg Lys Ala Gln         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Ala Leu Asp Val Gly Trp Leu Val Phe Asp Il - #e Thr Val Thr Ser Asn         #               255                                                           - His Trp Val Ile Asn Pro Gln Asn Asn Leu Gl - #y Leu Gln Leu Cys Ala         #           270                                                               - Glu Thr Gly Asp Gly Arg Ser Ile Asn Val Ly - #s Ser Ala Gly Leu Val         #       285                                                                   - Gly Arg Gln Gly Pro Gln Ser Lys Gln Pro Ph - #e Met Val Ala Phe Phe         #   300                                                                       - Lys Ala Ser Glu Val Leu Leu Arg Ser Val Ar - #g Ala Ala Asn Lys Arg         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Lys Asn Gln Asn Arg Asn Lys Ser Ser Ser Hi - #s Gln Asp Ser Ser Arg         #               335                                                           - Met Ser Ser Val Gly Asp Tyr Asn Thr Ser Gl - #u Gln Lys Gln Ala Cys         #           350                                                               - Lys Lys His Glu Leu Tyr Val Ser Phe Arg As - #p Leu Gly Trp Gln Asp         #       365                                                                   - Trp Ile Ile Ala Pro Glu Gly Tyr Ala Ala Ph - #e Tyr Cys Asp Gly Glu         #   380                                                                       - Cys Ser Phe Pro Leu Asn Ala His Met Asn Al - #a Thr Asn His Ala Ile         385                 3 - #90                 3 - #95                 4 -       #00                                                                           - Val Gln Thr Leu Val His Leu Met Phe Pro As - #p His Val Pro Lys Pro         #               415                                                           - Cys Cys Ala Pro Thr Lys Leu Asn Ala Ile Se - #r Val Leu Tyr Phe Asp         #           430                                                               - Asp Ser Ser Asn Val Ile Leu Lys Lys Tyr Ar - #g Asn Met Val Val Arg         #       445                                                                   - Ser Cys Gly Cys His                                                             450                                                                       - (2) INFORMATION FOR SEQ ID NO:19:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 513 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Protein                                                         (B) LOCATION: 1..513                                                #/note= "PRE-PRO-BMP6 (HUMAN)"N:                                              -      (x) PUBLICATION INFORMATION:                                                     (A) AUTHORS: CELESTE,                                                         (C) JOURNAL: Proc. Natl - #. Acad. Sci. U.S.A.                                (D) VOLUME: 87                                                                (F) PAGES: 9843-9847                                                          (G) DATE: 1991                                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                - Met Pro Gly Leu Gly Arg Arg Ala Gln Trp Le - #u Cys Trp Trp Trp Gly         #                15                                                           - Leu Leu Cys Ser Cys Cys Gly Pro Pro Pro Le - #u Arg Pro Pro Leu Pro         #            30                                                               - Ala Ala Ala Ala Ala Ala Ala Gly Gly Gln Le - #u Leu Gly Asp Gly Gly         #        45                                                                   - Ser Pro Gly Arg Thr Glu Gln Pro Pro Pro Se - #r Pro Gln Ser Ser Ser         #    60                                                                       - Gly Phe Leu Tyr Arg Arg Leu Lys Thr Gln Gl - #u Lys Arg Glu Met Gln         #80                                                                           - Lys Glu Ile Leu Ser Val Leu Gly Leu Pro Hi - #s Arg Pro Arg Pro Leu         #                95                                                           - His Gly Leu Gln Gln Pro Gln Pro Pro Ala Le - #u Arg Gln Gln Glu Glu         #           110                                                               - Gln Gln Gln Gln Gln Gln Leu Pro Arg Gly Gl - #u Pro Pro Pro Gly Arg         #       125                                                                   - Leu Lys Ser Ala Pro Leu Phe Met Leu Asp Le - #u Tyr Asn Ala Leu Ser         #   140                                                                       - Ala Asp Asn Asp Glu Asp Gly Ala Ser Glu Gl - #y Glu Arg Gln Gln Ser         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Trp Pro His Glu Ala Ala Ser Ser Ser Gln Ar - #g Arg Gln Pro Pro Pro         #               175                                                           - Gly Ala Ala His Pro Leu Asn Arg Lys Ser Le - #u Leu Ala Pro Gly Ser         #           190                                                               - Gly Ser Gly Gly Ala Ser Pro Leu Thr Ser Al - #a Gln Asp Ser Ala Phe         #       205                                                                   - Leu Asn Asp Ala Asp Met Val Met Ser Phe Va - #l Asn Leu Val Glu Tyr         #   220                                                                       - Asp Lys Glu Phe Ser Pro Arg Gln Arg His Hi - #s Lys Glu Phe Lys Phe         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Asn Leu Ser Gln Ile Pro Glu Gly Glu Val Va - #l Thr Ala Ala Glu Phe         #               255                                                           - Arg Ile Tyr Lys Asp Cys Val Met Gly Ser Ph - #e Lys Asn Gln Thr Phe         #           270                                                               - Leu Ile Ser Ile Tyr Gln Val Leu Gln Glu Hi - #s Gln His Arg Asp Ser         #       285                                                                   - Asp Leu Phe Leu Leu Asp Thr Arg Val Val Tr - #p Ala Ser Glu Glu Gly         #   300                                                                       - Trp Leu Glu Phe Asp Ile Thr Ala Thr Ser As - #n Leu Trp Val Val Thr         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Pro Gln His Asn Met Gly Leu Gln Leu Ser Va - #l Val Thr Arg Asp Gly         #               335                                                           - Val His Val His Pro Arg Ala Ala Gly Leu Va - #l Gly Arg Asp Gly Pro         #           350                                                               - Tyr Asp Lys Gln Pro Phe Met Val Ala Phe Ph - #e Lys Val Ser Glu Val         #       365                                                                   - His Val Arg Thr Thr Arg Ser Ala Ser Ser Ar - #g Arg Arg Gln Gln Ser         #   380                                                                       - Arg Asn Arg Ser Thr Gln Ser Gln Asp Val Al - #a Arg Val Ser Ser Ala         385                 3 - #90                 3 - #95                 4 -       #00                                                                           - Ser Asp Tyr Asn Ser Ser Glu Leu Lys Thr Al - #a Cys Arg Lys His Glu         #               415                                                           - Leu Tyr Val Ser Phe Gln Asp Leu Gly Trp Gl - #n Asp Trp Ile Ile Ala         #           430                                                               - Pro Lys Gly Tyr Ala Ala Asn Tyr Cys Asp Gl - #y Glu Cys Ser Phe Pro         #       445                                                                   - Leu Asn Ala His Met Asn Ala Thr Asn His Al - #a Ile Val Gln Thr Leu         #   460                                                                       - Val His Leu Met Asn Pro Glu Tyr Val Pro Ly - #s Pro Cys Cys Ala Pro         465                 4 - #70                 4 - #75                 4 -       #80                                                                           - Thr Lys Leu Asn Ala Ile Ser Val Leu Tyr Ph - #e Asp Asp Asn Ser Asn         #               495                                                           - Val Ile Leu Lys Lys Tyr Arg Asn Met Val Va - #l Arg Ala Cys Gly Cys         #           510                                                               - His                                                                         - (2) INFORMATION FOR SEQ ID NO:20:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 97 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Protein                                                         (B) LOCATION: 1..97                                                 #/label= Generic-Seq-7FORMATION:                                              #"wherein each Xaa is independently selected                                  #group of one or more specified amino acids                                                  as define - #d in the specification."                          -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                - Leu Xaa Xaa Xaa Phe Xaa Xaa Xaa Gly Trp Xa - #a Xaa Xaa Xaa Xaa Xaa         #                15                                                           - Pro Xaa Xaa Xaa Xaa Ala Xaa Tyr Cys Xaa Gl - #y Xaa Cys Xaa Xaa Pro         #            30                                                               - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asn His Al - #a Xaa Xaa Xaa Xaa Xaa         #        45                                                                   - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Cys Cys Xaa Pro         #    60                                                                       - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Leu Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa         #80                                                                           - Val Xaa Leu Xaa Xaa Xaa Xaa Xaa Met Xaa Va - #l Xaa Xaa Cys Xaa Cys         #                95                                                           - Xaa                                                                         - (2) INFORMATION FOR SEQ ID NO:21:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 102 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Protein                                                         (B) LOCATION: 1..102                                                #/label= Generic-Seq-8FORMATION:                                              #"wherin each Xaa is independently selected                                   #group of one or more specified amino acids                                                  as define - #d in the specification."                          -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                - Cys Xaa Xaa Xaa Xaa Leu Xaa Xaa Xaa Phe Xa - #a Xaa Xaa Gly Trp Xaa         #                15                                                           - Xaa Xaa Xaa Xaa Xaa Pro Xaa Xaa Xaa Xaa Al - #a Xaa Tyr Cys Xaa Gly         #            30                                                               - Xaa Cys Xaa Xaa Pro Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Asn His Ala         #        45                                                                   - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa         #    60                                                                       - Xaa Cys Cys Xaa Pro Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Leu Xaa Xaa         #80                                                                           - Xaa Xaa Xaa Xaa Xaa Val Xaa Leu Xaa Xaa Xa - #a Xaa Xaa Met Xaa Val         #                95                                                           - Xaa Xaa Cys Xaa Cys Xaa                                                                 100                                                               - (2) INFORMATION FOR SEQ ID NO:22:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 102 amino                                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Protein                                                         (B) LOCATION: 1..102                                                #/label= OPX) OTHER INFORMATION:                                              #"WHEREIN EACH XAA IS INDEPENDENTLY SELECTED                                  #GROUP OF ONE OR MORE SPECIFIED AMINO ACIDS                                                  AS DEFINE - #D IN THE SPECIFICATION (SECTION I)"               -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                - Cys Xaa Xaa His Glu Leu Tyr Val Xaa Phe Xa - #a Asp Leu Gly Trp Xaa         #                15                                                           - Asp Trp Xaa Ile Ala Pro Xaa Gly Tyr Xaa Al - #a Tyr Tyr Cys Glu Gly         #            30                                                               - Glu Cys Xaa Phe Pro Leu Xaa Ser Xaa Met As - #n Ala Thr Asn His Ala         #        45                                                                   - Ile Xaa Gln Xaa Leu Val His Xaa Xaa Xaa Pr - #o Xaa Xaa Val Pro Lys         #    60                                                                       - Xaa Cys Cys Ala Pro Thr Xaa Leu Xaa Ala Xa - #a Ser Val Leu Tyr Xaa         #80                                                                           - Asp Xaa Ser Xaa Asn Val Xaa Leu Xaa Lys Xa - #a Arg Asn Met Val Val         #                95                                                           - Xaa Ala Cys Gly Cys His                                                                 100                                                               - (2) INFORMATION FOR SEQ ID NO:23:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 4 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Cleavage-sit - #e                                               (B) LOCATION: 1..4                                                  #/note= "PROTEOLYTIC CLEAVAGE SITE"                                           -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                - Arg Xaa Xaa Arg                                                             __________________________________________________________________________

What is claimed is:
 1. A method for evaluating the disease state of atissue or body fluid, comprising:comparing in a tissue or body fluidsample a detected amount to an expected amount of an antibody capable ofbinding to a soluble morphogen complex, said complex comprising adimeric protein having an amino acid sequence selected from the groupconsisting of:(a) a sequence having at least 70% homology with theC-terminal seven-cysteine skeleton of human OP-1, residues 330-431 ofSEQ. ID NO. 2, and (b) Generic Sequence 7, SEQ. ID NO. 20; said dimericprotein being in non-covalent association with a morphogen pro region orfragment thereof.
 2. A method for evaluating the efficacy of a therapyfor regenerating lost or damaged tissue in a mammal, the methodcomprising:comparing in a tissue or body fluid sample a detected amountto an expected amount of an antibody capable of binding to a solublemorphogen complex, said complex comprising a dimeric protein having anamino acid sequence selected from the group consisting of:(a) a sequencehaving at least 70% homology with the C-terminal seven-cysteine skeletonof human OP-1, residues 330-431 of SEQ. ID NO. 2, and (b) GenericSequence 7, SEQ. ID NO. 20; said dimeric protein being in non-covalentassociation with a morphogen pro region or fragment thereof.
 3. A methodfor diagnosing a tissue disorder in a mammal, the methodcomprising:comparing in a tissue or body fluid sample a detected amountto an expected amount of an antibody capable of binding to a solublemorphogen complex, said complex comprising a dimeric protein having anamino acid sequence selected from the group consisting of:(a) a sequencehaving at least 70% homology with the C-terminal seven-cysteine skeletonof human OP-1, residues 330-431 of SEQ. ID NO. 2, and (b) GenericSequence 7, SEQ. ID NO. 20; said dimeric protein being in non-covalentassociation with a morphogen pro region or fragment thereof.
 4. Themethod of claim 1 wherein said method is an immunoassay.
 5. The methodof claim 1, 2 or 3 wherein said pro region is derived from a bonemorphogenic protein comprising at least 100 amino acids sharing at least70% amino acid sequence homology with residues 330-431 of SEQ. ID NO. 2(human OP-1).
 6. The method of claim 1, 2 or 3 wherein said antibody iscapable of distinguishing soluble morphogen complex from insolublemorphogen and non-morphogen proteins in a body fluid or tissue sample.7. The method of claim 3 wherein said tissue disorder is a bone tissuedisorder.
 8. The method of claim 7 wherein said bone tissue disorder isselected from the group consisting of osteosarcoma, osteoporosis, andPaget's disease.